Sexually Dimorphic Expression and Regulation of Sfswap in The

SEXUALLY DIMORPHIC EXPRESSION AND REGULATION OF SFSWAP IN THE

DEVELOPING MOUSE CORTEX AND HIPPOCAMPUS

A THESIS

Presented to the University Honors Program

California State University, Long Beach

In Partial Fulfillment

of the Requirements for the

University Honors Program Certificate

Aaron Ridder

Spring 2016

I, THE UNDERSIGNED MEMBER OF THE COMMITTEE,

HAVE APPROVED THIS THESIS

SEXUALLY DIMORPHIC EXPRESSION AND REGULATION OF SFSWAP IN THE

DEVELOPING MOUSE CORTEX AND HIPPOCAMPUS

Aaron Ridder

Houng-Wei Tsai, Ph.D. (Thesis Advisor) Biological Sciences Department

California State University, Long Beach

Spring 2016

ABSTRACT

SEXUALLY DIMORPHIC EXPRESSION AND REGULATION OF SFSWAP IN THE

DEVELOPING MOUSE CORTEX AND HIPPOCAMPUS

Aaron Ridder

May 2016

The cortex and hippocampus are important for the control of cognitive and social behaviors, many of which are sexually dimorphic. However, the molecular mechanisms underlying these functional differences between the sexes have not been discovered yet. Since increasing evidence points to defects in regulation of splicing implicated in a variety of neurological diseases and mental illnesses served by the cortex and hippocampus, I speculate similar molecular mechanisms might be involved in sexual dimorphism in these brain regions.

Along with this, in rodents and humans, there is a perinatal rise in testosterone in males that masculinizes neural structures in the brain during development. This rise in T may regulate a variety of functional genes giving rise to observable sex differences in expression and behavior.

Splicing factor suppressor of white apricot (Sfswap) is one of the novel splicing factors highly expressed throughout the mouse brain, including the cortex and the hippocampus and plays an important role in alternative splicing. I hypothesized that Sfswap expression in the developing mouse cortex/hippocampus was sexually dimorphic and that it is regulated by this perinatal rise in testosterone, which might be critical for establishing differential neural structures and behaviors between the sexes. To test my hypothesis, I used reverse transcription with

i quantitative polymerase chain reaction (RT-qPCR) and immunoblotting to measure mRNA and

protein levels of Sfswap in the cortex/hippocampus of male and female mice collected on the day

of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. Results showed that

SFSWAP protein levels in the female cortex/hippocampus were higher than males on PN7, PN14

and PN21. To test the regulation of Sfswap, injections of testosterone proprionate were

administered prenatally and postnally. Female SFSWAP expression in mice treated postnally were significantly lowered to that of the male expression. SFSWAP expression in Tfm mice, lacking the receptor for testosterone, were also analyzed. Preliminary data showed that Tfm males had similar expression to that of females, indicating a selective decrease in expression due to neonatal testosterone. Taken together, I conclude that in the developing mouse cortex/hippocampus, SFSWAP expression is sexually dimorphic and is regulated by testosterone

through a variety of mechanisms. Our findings indicate that sexually dimorphic SFSWAP

expression might help formulate sex differences in brain structures and behaviors.

ii ACKNOWLEDGEMENTS

I would like to acknowledge my friends, family, and God for helping me get through this thesis. I would have never made it without them. I would especially like to thank Dr. Houng-Wei

Tsai for his continual support and encouragement through it all. For all my fans, paper and real, this is for you.

iii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS……………………………………………………………………....iii

LIST OF TABLES……………………………………………………………………………….vi

LIST OF FIGURES……………………………………………………………………………...vii

LIST OF ABBREVIATIONS…………………………………………………………………….ix

CHAPTER

1. INTRODUCTION……………………………………………………………………...1

Sex Chromosomes and Chromosomal Sex………………………………………..1

Sex Differences in the Cortex/Hippocampus……………………………………...2

Sex Differences due to Splicing…………………………………………………...3

Splicing Factor, Suppressor of White-Apricot Homolog…………………………4

Hypothesis…………………………………………………………………………6

2. MATERIALS AND METHODS……………………………………………………….7

Animals……………………………………………………………………………7

Experimental Design……………………………………………………………....7

RNA Extraction and cDNA synthesis……………………………………………..8

PCR primer testing………………………………………………………………...8

RT-qPCR………………………………………………………………………...10

Protein Extraction………………………………………………………………..10

Immunoblotting……...………………………………………………………...…11

Testosterone Propionate Treatments prenatally and postnatally………………...11

Statistical Analysis……………………………………………………………….12

iv 3. RESULTS……………………………………………………………………………..13

Experiment 1: Developmental Expression of Sfswap…………………………...13

Experiment 2: Effects of Testosterone Propionate on Expression of

SFSWAP…………………………………………………………………………15

Experiment 3: Effects of Testicular Feminization Mutation on Expression of

SFSWAP…………………………………………………………………………18

4. DISCUSSION…………………………………………………………………………19

Sexually Dimorphic SFSWAP Expression during Development………………..19

Potenetial Isoform of SFSWAP………………………………………………….22

Difference in mRNA and Protein Expression of Sfswap as age increases………23

Effects of Testosterone Proprionate on Expression of SFSWAP………………..23

Effects of Testicular Feminization on Expression of SFSWAP…………………24

Hypothetic Working Model on the Regulation of SFSWAP…………………….25

APPENDICES…………………………………………………………………………………...27

A. TABLES………………………………………………………………………………28

B. FIGURES…………………………………………………………………………..…33

REFERENCES…………………………………………………………………………………..43

v LIST OF TABLES

TABLE Page

1. List of Oligonucleotide Primers used for RT-PCR and RT-qPCR………………………29

2. Mouse weights, tissue weights, and protein yield used for protein analysis.……………30

3. Mouse weights, tissue weights, and RNA yield used for mRNA analysis ………...……31

4. Ano-Genital Distance (AGD) …………………………………………………………...32

vi LIST OF FIGURES

FIGURE Page

1. Expression of mRNA Sfswap at the developmental ages……………………..…………34

2. Representative Immunoblotting of SFSWAP protein in male (M) and female

(F) on age………………………………………………………………………...35

3. Expression of SFSWAP 104kDa protein in the cortex/hippocampus of male

and female mouse pups on the day of birth (PN0), and 7 (PN7), 14

(PN14), and 21 (PN21) after birth……………………………………………….36

4. Expression of SFSWAP 100kDa protein in the cortex/hippocampus of male

and female mouse pups on the day of birth (PN0), and 7 (PN7), 14

(PN14), and 21 (PN21) after birth………………..…………………………...…37

5. Expression of SFSWAP protein in the cortex/hippocampus comparing age

in mouse pups on the day of birth (PN0) and 21 (PN21) days after birth…….…38

6. Relative expression levels of Sfswap in the cortex/hippocampus of prenatal

male (M) and female (F) mice treated with vehicle (V) and testosterone

proprionate (TP) ………………………………………………………………....39

7. Relative expression levels of Sfswap in the cortex/hippocampus of postnatal

male (M) and female (F) mice treated with vehicle (V) and testosterone

proprionate (TP) …………………………………………………………………40

8. Relative expression levels of SFSWAP in the cortex/ hippocampus of testicular

feminization mutation (Tfm) mice as well as wild-type male (M)

and female (F) littermates at PN21………………………………………………41

vii

9. Hypothetic working model that illustrates the development of

sexually dimorphic SFSFWAP expression in the developing

mouse cortex/hippocampus……………………………………………………...42

viii

LIST OF ABBREVIATIONS

AGD Ano-genital distance

ANOVA Analysis of variance

AR Androgen receptor

AVPV Anteroventral periventricular nucleus

bp base pairs

Ct Cycle at threshold in qPCR

E16 Embryonic day 16 (16 days after a mating plug is seen)

E18 Embryonic day 18 (18 days after a mating plug is seen)

ER Estrogen Receptor

H3 Histone H3

H3K9/14Ac Acetylated ninth or fourteenth lysine of H3

H3K9Me3 Trimethylated ninth lysine of H3

kDa Kilodalton

MGI Mouse Genome Informatics

NCBI National Center for Biotechnology Information

PN0 Postnatal day 0 (the day of birth)

PN7 Postnatal day 7 (7 days after birth)

PN14 Postnatal day 14 (14 days after birth)

PN21 Postnatal day 21 (21 days after birth)

PN23 Postnatal day 23 (23 days after birth)

SD Standard Deviation

ix SDN Sexually Dimorphic Nucleus

SEM Standard error of the mean

T Testosterone

TP Testosterone proprionate

T3 Triiodothyronine

CHAPTER 1

INTRODUCTION

Sex differences in normal brain function and behavior are similar in many species, including

humans (Breedlove and Hampson 2002; Jazin and Cahill 2010; McCarthy and Arnold 2011).

Gender differences are also noted in the prevalence and symptomatology of many neurological

diseases and mental illnesses, such as depression, schizophrenia, and autism (Baron-Cohen et al.,

2009; Deng et al., 2010; Greer and McCombe 2011). Understanding of the mechanisms that

establish sexual dimorphism in behaviors and underlying neural structures will help us identify

the processes causing gender-specific susceptibility to different diseases, which may assist in the

development of new treatments for these sex-biased disorders.

Sex Chromosomes and Chromosomal Sex

Early development in mice and humans begins with the complement of the two sex

chromosomes, X and Y. Females (XX) and males (XY) differ in development due to this

variation in the complement. The Sry gene in the sex determining region of the Y chromosome

produces a transcription factor expressed in the male undifferentiated gonads, leading to

formation of the testis (Kopsida et al., 2009). During the late gestation, embryonic days 16 and

17 (E16 and E17) and immediately after birth (denoted as the critical period), a transient rise in

circulating testosterone (T) released by the developing testes formulates sex differences in brain

structures and behaviors, laying the foundation which is known as the organizational effect

(Motelica-Heino et al., 1988; Phoenix et al., 1959). This organizational effect induces permanent changes in structure, such as in the formation and development of sexually dimorphic nucleus of

the hypothalamus (Morris et al., 2004). This differs to activational effect during puberty, where

the change is not permanent but rather a transient effect, such as when estrogen and progesterone

“activate” lordosis in female rats (Powers 1970). Using mice and rats as the models,

neuroendocrinologists have demonstrated that this perinatal rise in T might act (1) directly on

androgen receptor (AR) and/or (2) indirectly on estrogen receptors (ERs) via estradiol that is

synthesized locally by aromatase to masculinize a variety of neural structures and circuitry in the

hypothalamus and amygdala responsible for male-specific behaviors (Davis et al., 1996; Forger

2009; Hines 2006). These steroid receptors are abundant in the rodent brain, including the cortex and hippocampus (Ivanova and Beyer 2000; Kerr et al., 1995), and are important for sex differences in many cognitive and social behaviors served by these brain regions, such as learning and memory (Frick and Gresack Dec 2003; Sutcliffe et al., 2007). Functionally, this perinatal rise in T during the critical period leads to masculinization of sexually dimorphic neural circuitry and behavior (Phoenix et al., 1959). However, the molecular mechanisms underlying the action of sex steroids on expression of downstream genes, as relevant to these sex differences, still remain unclear.

Sex Differences in the Cortex/Hipocampus

The cortex/hippocampus is a particular region of the brain subject to a variety of sex

differences. The cerebral cortex/hippocampus are involved in the control of social behaviors and

cognitive functions, many of which are sexually dimorphic (Swaab et al., 2001). Sex differences

in the cortex/ hippocampus have since been established. Structural, and therefore functional,

differences in male and female cortex/hippocampus have also been reported. The cortex and

hippocampus are also found to be sexually dimorphic in prevalence and symptomatology of

many neurological diseases and mental illnesses (Ngun et al., 2011). Humans suffering from

2 autism have been found to display a greater thickness in their cortex (Carper et al., 2002; Doyle-

Thomas et al., 2013). Autism is a male biased neurological disorder characterized by deficiencies in social and interpersonal behavior (Baron-Cohen et al., 2011). Alzheimer’s disease, a disease more found in women, is localized to the hippocampus and impairs learning and memory (Mu and Gage 2011). In these sex specific neurological disorders, one sex is protected more highly than another. Males have a higher perceptibility than females to autism and females have a higher chance than males to develop Alzheimer’s disease. Continued identification and characterization of regulation in key regions of the brain can lead to developments of treatments to alleviate these diseases all together.

Hormonal influences have been shown to have a strong determining factor in the sex differences of the cortex/hippocampus. Due to hormonal influences rather than sex chromosome complement, cortical thickness in adult mice are larger in males than in females. (Markham et al., 2003). Due to T exposure, the rat hippocampus exhibits increased neurogenesis during the first week of life (Merkley et al., 2014). Since T has been known to influence neural functions such as cell survival, maintenance, and proliferation, this same regulation is likely to occur in a variety of sex specific genes leading to the formation of observable sex differences in the cortex/hippocampus.

Sex Differences due to Splicing

Besides hormonal regulation, formation of sex differences can be due to a variety of functions such as splicing. In Drosophila, male-specific fruitless (fru) protein expression regulated by alternative splicing has been shown to control male sexual behavior (Demir and

Dickson 2005). Male courtship and sexual orientation in Drosophila are products of the fruitless

(fru) gene, which is spliced in both the male and the female. Regulation of alternative splicing of

this gene causes these downstream effects in males, and a lack of these behaviors in females.

However, a similar mechanism has not been demonstrated in mice or humans. Splicing,

therefore, may be critical in understanding complex sexually dimorphic behaviors and the

formation of these sex differences.

Numerous neurological disorders and mental illnesses associated with splicing defects

emphasizes the importance of alternative splicing in regulating brain function (Dredge et al.,

2001; Poulos et al., 2011). Many of these disorders display sex differences in the prevalence and

symptomatology served by the cerebral cortex and hippocampus. Splicing regulation is known to

control the expression of various specific genes in the mouse cortex giving rise to a selection of

neurological disorders associated with splicing (Dillman et al., 2013; Vuong et al., 2016). Sex

differences in these splicing factors can establish sexually dimorphic development of cortical

structure and function. In the hippocampus, sexually dimorphic expression of two splicing

factors, Psf and Srp20, affect the formation of long-term memory which is critical in Alzheimer’s

disease (Antunes-Martins et al., 2007). Alternative splicing, therefore, has been known to

regulate sex differences in males and females.

Splicing Factor, Suppresor of White-Apricot Homolog

Due to a lack of well-characterized dimorphic biomarkers in the cortex/hippocampus, a

preliminary study using gene expression microarrays to identify genes differentially transcribed in the neonatal mouse cortex between the sexes was needed (Bonthuis et al., 2010; Corbier et al.,

1992; McCarthy and Ball 2008; Rissman 2008). One such gene, Splicing Factor, Suppressor of

White-Apricot Homolog (Drosophila) (Sfswap) was one of the many potential genes in the

developing mouse cortex/hippocampus that is differentially expressed between the sexes. Since alternative splicing has been implicated in the process of sexual differentiation, this gene, which is recognized for its alternative splicing throughout the brain, may be critical for the development of sexually dimorphic functions.

Sfswap encodes a human and mouse homolog of Drosophila splicing regulatory protein. This splicing factor regulates the splicing of several genes. Specifically, Sfswap represses MAPT/Tau exon 10 splicing (Kar et al., 2005). Errors in alternative splicing of tau can lead to neurological degenerative disorders such as frontotemporal dementia. Sfswap has been reported to also enhance the splicing of fibronectin and CD45, proteins used ubiquitously for wound healing and immune function (Sarkissian et al., 1996). CD45 expression has been shown to have a sex difference in women with autoimmune hematolic disorders, serving a critical function in women’s health (Mylvaganam et al., 1989). In Drosophia, Sfswap has even been shown to autoregulate its own mRNA through control of its first two introns (Denhez and Lafyatis 1994).

Sfswap has been shown to be regulated hormonally as well. Sfswap is under thyroid hormone

control in the rat brain during the critical period of neuronal differentiation (Cuadrado et al.,

1999). Thyroid hormone plays a critical role in brain maturation and prevention of neurological

deficits and mental retardation (Dickson et al., 1987; Dussault and Ruel 1987). Treatments of

thyroid caused a decrease in Sfswap expression throughout the rat brain except the cerebellum.

Furthermore, in hypothyroid conditions, conditions lacking thyroid hormone, Sfswap levels were

abnormally high. Triiodothyronin (T3) is known to regulate gene transcription through

activation, repression and transcriptional interference of other transcription factors near the

genome, and even post transcriptionally (Fraboulet et al., 1996; Puymirat et al., 1995). This

direct influence on transcriptional regulation, implicates the nature of the downstream effects of

Sfswap and leads to the speculation of this gene being regulated similarly by other hormones.

Hypotheses

Taken together, I hypothesize that Sfswap is sexually dimorphic and that it might be regulated by the perinatal rise in T, leading to the development of distinct neural functions and behaviors between the sexes. In this study, I use a mouse model to analyze Sfswap for sex differences. In addition to 95% similarity in genome, mouse and human Sfswap mRNA reveal

splice junctions of these regulated introns to be precisely conserved, implicating a similar

process between these species.

To test this hypothesis, I analyzed the expression of Sfswap mRNA and protein between the

sexes on the developmental postnatal days 0 (PN0), 7 (PN7), 14 (PN14), and 21 (PN21). To

further test my hypothesis, I examined the effects of prenatal T and postnatal T on Sfswap

expression to determine whether it is regulated by T. In addition, mice with the testicular

feminization mutation (Tfm) were used to analyze the expression of SFSWAP in mice without a

functional androgen receptor.

CHAPTER 2

MATERIALS AND METHODS

Animals

Adult male and female C57BL/6J mice (Mus musculus) were purchased from the Jackson

Laboratory (Bar Harbor, ME) and housed in the California State University, Long Beach

(CSULB) Animal Care Facility. Mice were kept on a 12/12 L/D cycle, lights on at 0600. Food

(Harlan Teklad Mouse/Rat Diet #7012) and water were provided ad libitum. Adult female mice were paired with fertile males, and checked each morning for the presence of mating plugs; the day the plug was found was designated as embryonic day 0 (E0). Males were removed and unpaired when pregnancy was reaching completion (E14-E17).

Testicular Feminization Mice (Tfm) were purchased from Jackson Laboratory carrying the

Tfm allele. These were bred with C57BL/6J mice and kept in conditions mentioned above. Upon weaning at 21 days old, mice were ear punched and genotyped using a modified polymerase chain reaction (PCR) (Fernandez et al., 2003) Based on the presence or absence of the Sry gene found only on the Y chromosome, this technique was able to differentiate between the Tfm and wt allele for the AR, along with male versus female. All experimental procedures were approved by CSULB Animal Use and Care Committee and performed according to Association for

Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.

Experimental Design

Male and female pups were sacrificed by rapid decapitation on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. Their brains were immediately removed and dissected into various regions. The cortex along with the hippocampus were dissected out and stored at -80˚C until processed either for RNA or Protein extraction. Besides examining the

presence of pigmentation (males) in the anogenital regions, the gonads and reproductive tracts of

these pups were also dissected out to confirm their sexes.

RNA Extraction and cDNA synthesis

Total RNA was extracted from the mouse cortex/hippocampus using RNeasy® Lipid Tissue

kits (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol. RNA quality and

concentration of individual samples was determined using Bio-Rad SmartSpec Plus

spectrophotometer (Hercules, CA) as the samples were diluted (1:50) in 10 mM Tris (pH = 7.5).

Absorbance at 260 nm (A260), 280 nm (A280), and 320 nm (A320) was measured. RNA

concentration was calculated with A260, and purity was determined by the A260/A280 ratio

(between 1.9 and 2.1). The cDNA samples were synthesized from 1 μg extracted RNA in 20-μl

reactions using the Bio-Rad iScript cDNA Synthesis Kit. The reverse transcription was

performed at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min using a Bio-Rad

MyCycler™ thermocycler.

PCR primer testing

The oligonucleotide primers of Sfswap and two house-keeping genes, Rpl13a and Actb, were designed and then checked for suitability and sequence specificity using the Primer3 program

(http://primer3.wi.mit.edu/) and NCBI’s Primer-BLAST program

(http://www.ncbi.nlm.nih.gov/tools/primer-blast/) against RefSeq RNA, respectively.

The primers purchased from Eurofins MWG Operon (Huntsville, AL) were first tested in

RT-PCR assays for 40 cycles using GoTaq® Hot Start Green Master Mix (Promega, Madison,

WI) according to the manufacturers' protocol. For each primer pair, PCR tests were performed at an annealing temperature of 2 °C below the lower melting temperature of the forward and

reverse primers as indicated by Eurofins MWG Operon. PCR products were separated on a 1.5%

agarose gel and visualized by ethidium bromide staining. All three primer sets used in this study

produce single PCR products with the correct size in the positive control (with cDNA) as

calculated, but no product in the negative control (no cDNA).

Next, cDNA stocks were serially diluted from 1:8 to 1:1,024. Each qPCR reaction containing

4 μl of cDNA, 1 μl each of forward and reverse primers (840 nM), and 6 μl of ABsolute™

QPCR SYBR® Green Mix (Waltham, MA) was performed on an Agilent Stratagene®

MX3000P™ qPCR system with MxPro QPCR software (Santa Clara, CA). qPCR amplification

was conducted with an initial denaturation/polymerase-activation step at 95 °C for 15 min, 40 cycles of 15 s at 95 °C, 1 min at the annealing temperature, and 30 s at 72 °C, and a final dissociation step with 1 min at 95 °C, 30 s at 60 °C, and 30 s at 95 °C. Cycle of thresholds (Cts) were analyzed by linear regression with log base 2 of the dilution as the predictor and Ct values as the response. All three sets of primers we used show the average R2 of the regression of 0.98,

Ct values at the 1:16 dilution above 30, and the dissociation curve with single peaks. A dissociation curve with a single peak confirmed that a single product of the same size was correctly amplified by qPCR. The primers of Sfswap, Rpl13a, and Actb pass the primer tests and are considered suitable for PCR quantification. The annealing temperatures used and sizes of their PCR products are listed in Table 1.

RT-qPCR

RT-qPCR reactions for Sfswap, Rpl13a, and Actb gene expression quantification were run

under the same conditions as described in the primer dilution tests above, with 4 μl of 1:16

diluted cDNA. Two replicate reactions for each sample were run, and their Ct values were

averaged for further analysis. For a given sample unnormalized relative expression values for

gene expression were calculated using Pfaffl’s method against a female baseline (PN0-F) for all

cases (Pfaffl 2001); this female baseline was established by averaging the Ct values of all PN0

female. Relative expression values of Sfswap gene were divided by the geometric mean of the

relative expression values of Rpla13a and Actb genes to yield normalized relative expression

values (Vandesompele et al., 2002). Values more than two standard deviations from their group

mean were trimmed to two standard deviations from the group mean in the same direction.

Protein Extraction

The mouse cortex/hippocampus were homogenized in T-PER buffer (Thermo Fisher

Scientific/Pierce Biotechnology, Rockford, lL) with protease inhibitor cocktail (Sigma, St. Louis,

MO) and PMSF (1 mM) Brain tissues were homogenized by drawing and ejecting 15-20 times

through the needles (20G) of sterile syringes, followed by centrifugation at 10,000 g for 5 min at

4 °C. The supernatant was separated from the pallet and then stored at -80°C freezer. The lysate

protein concentrations of individual samples were determined by bicinchoninic acid (BCA)

assays using the Thermo Scientific Pierce™ BCA Protein Assay Kit (Rockford, IL)

Immunoblotting

Protein samples of 20 μg each were subjected to electrophoresis on 8% sodium dodecyl

sulfate polyacrylamide gel electrophoresis (SDS-PAGE) then transferred to nitrocellulose membranes using the BioRad Trans-Blot Turbo Transfer System (Bio-Rad). The membranes

were rinsed in Tris-buffered saline with 0.1% Tween-20 (TBST) and then blocked in TBST with

10% milk overnight. After blocking, blots were rinsed with TBST and then incubated with the

primary antibody against SFSWAP protein (Prod# HPA039362, Lot#R37173, Sigma) (1:2,000)

10 for 1 hr at room temperature. After rinsing, blots were then incubated for 1 hr in a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:10,000). After rinses, SFSWAP protein bands was detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce) and imaged by Protein Simple Fluor Chem E (ProteinSimple, ).After rinsing, the same blots were incubated with the primary antibody against β-actin (1:200,000 in TBST) (Cat#A5441), followed by the HRP-conjugated anti-mouse IgG antibodies (1:50,000) and detection of chemiluminescence.

The intensities of Sfswap and β-actin proteins were measured and analyzed with Alpha View

(Protein Simple). To normalize each individual sample, the densities of Sfswap protein were normalized with the density β-actin detected by western blot. All the samples from each experiment could not be run on the same gel. Therefore, to reduce inter-gel variation, the normalized Sfsawp protein levels in individual samples were standardized to the mean level of females in the same developmental stage (sex effect), the PN0 group of the same sex (age effect) or the female vehicle (treatment effect) and each sample was expressed as a fold of the female,PN0, or vehicle female (set as 1 fold).

Testosterone Proprionate Treatments prenatally and postnatally

Male and female mice were injected with either vehicle (0.05 ml sesame oil) or testosterone propionate (100 µg in 0.05 ml sesame oil) subcutaneously daily at 0900 for three days. Prenatal treatments began on E16 and continued until birth. Postnatal treatments began on PN21 and continued until PN23. Following treatments, collection of the cortex/hippocampus were done and either prepared for RNA or protein extraction for analysis.

Statistical Analysis

Relative mRNA levels of Sfswap gene were analyzed by two-way ANOVA where the two

factors were sex and age, accompanied by Tukey’s HSD post-hoc tests where appropriate. A p-

value <0.05 was considered statistically significant.

SFSWAP protein data was analyzed using t-test with a significance value of p < 0.05.

Relative SFSWAP data were analyzed with sex or age as the single factor. Treatments with

testosterone proprionate data were analyzed by two-way ANOVA where the two factors were

sex and treatment, accompanied by Tukey’s HSD post-hoc tests where appropriate. A p-value

<0.05 was considered statistically significant.

The data from individual groups were first tested for normality using Anderson-Darling test, and if passed, the data were subjected to two-tailed, t-test. In the case of significant deviations from normality, measurements were log transformed, followed by t-test. If transformation still failed to normalize the data, the data were subjected to non-parametric Mann-Whitney test.

CHAPTER 3

RESULTS

Experiment 1: Developmental Expression of Sfswap

Mouse, Tissue, Protein Yield, RNA Yield and Ano-genital Distance

Measurements of mouse body weight at PN0 ( =0.72) and PN21 ( =0.27) showed no

significant sex difference. There was a female bias in body𝑃𝑃 weight on PN7𝑃𝑃 ( =0.03) and a male

bias in body weight on PN14 ( =0.02). Overall weight of cortex/hippocampus𝑃𝑃 during the

developmental ages (PN0 =0.40;𝑃𝑃 PN7 =0.39; PN14 =0.83; PN21 =0.67) showed no

significant differences between𝑃𝑃 male and𝑃𝑃 female brain samples𝑃𝑃 (Table 𝑃𝑃2). Protein yield had no

sex differences on PN0 ( =0.81), PN7 ( =0.72), PN14 ( =0.32), or PN21 ( =0.16). Overall

mRNA, for all ages during𝑃𝑃 development,𝑃𝑃 body weight (PN0𝑃𝑃 =0.98; PN7 =0.29;𝑃𝑃 PN14 =0.46;

PN21 =0.46), tissue weight (PN0 =0.51; PN7 =0.70; PN14𝑃𝑃 =0.75; PN21𝑃𝑃 =0.38), and𝑃𝑃

RNA Yield𝑃𝑃 (PN0 =0.36; PN7 =0.99;𝑃𝑃 PN14 =0.658;𝑃𝑃 PN21 𝑃𝑃=0.652) showed𝑃𝑃 no significant

sex differences (Table𝑃𝑃 3). Analysis𝑃𝑃 of ano-genital𝑃𝑃 distance (AGD)𝑃𝑃 on the mice collected

indicated a significant male bias on the days of 7 ( =0.007), 14 ( =0.001), and 21 ( =0.003)

(Table 4). 𝑃𝑃 𝑃𝑃 𝑃𝑃

Sfswap mRNA Expression

Sfswap had a significant sex difference at the age of PN7 ( =0.029), males expressing

higher levels than females. There was no significant sex difference𝑃𝑃 at the ages of PN0 (

=0.982), PN 14 ( =0.892), and PN21 ( =0.610) (Figure 1). Sfswap expressed a significant𝑃𝑃 age difference from PN0𝑃𝑃 to PN21. There was𝑃𝑃 a decrease of expression from PN0 on PN14 and PN21

( <0.001) and from PN7 on PN14 ( =0.054) and PN21 ( = 0.002). This indicated an overall

𝑃𝑃 𝑃𝑃 𝑃𝑃

significant decrease in Sfswap expression from PN0 to PN21. PN0 expressed around, on average,

2 times more highly Sfswap than PN21 mice.

SFSWAP Expression

SFSWAP protein is expressed in the cortex and hippocampus as shown in the

representative blot (Figure 2). Observed are multiple bands of significant intensity, many of

which may be potential isoforms of SFSWAP. Of those observed include: the full-length

SFSWAP with a molecular weight of 104 kDa and molecular weights at 150kDa, 100kDa, and

74kDa. Besides the full-length SFSWAP of 104kDa, the 100kDa band was also analyzed.

SFSWAP Expression of Full-Length SFSWAP Protein (104kda) During Developmental Ages

Full-length SFSWAP (104kDa) was expressed at significantly higher levels in females than in males on PN7, PN14, and PN21. Full-length SFSWAP protein levels at birth, PN0, in female

controls (1.01 ± 0.04, =6) were 6% greater than males (0.94 ± 0.06, =7) ( = 0.35). However, this difference dramatically𝑛𝑛 increases with female expression (1.00 ± 𝑛𝑛0.08, 𝑃𝑃=6) on PN7 being

52% greater than males (0.48 ± 0.06, =7) ( = 0.0002).This difference continues𝑛𝑛 on PN14

(male 0.75 ± 0.06, =7; female 1.00 ±𝑛𝑛 0.05, 𝑃𝑃=6) ( = 0.006) and PN21 (male 0.72 ± 0.05, =7; female 1.00 ± 0.09,𝑛𝑛 =6) ( = 0.008) (Figure𝑛𝑛 3). 𝑃𝑃 𝑛𝑛

SFSWAP Expression𝑛𝑛 of Potential𝑃𝑃 Isoform of SFSWAP Protein (100kda) During Developmental

Ages

Potential isoform of 100kDa Sfswap was expressed at significantly higher levels in females than in males on PN7 and PN14 but not the PN0 or PN21 (Figure 4). PN0 females (1.00

± 0.12, =6) had similar levels to males (0.93 ± 0.05, =7) ( = 0.33). Mice on PN7 and PN14 both had𝑛𝑛 significant differences in male and female expression𝑛𝑛 𝑃𝑃 showing a female bias (PN7: female 1.00 ± .13, =6; male 0.64 ± .10, =7 ( = 0.05)) (PN14: female 1.00 ± 0.04, =6;

𝑛𝑛 𝑛𝑛 𝑃𝑃 𝑛𝑛 14

male 0.81 ± 0.04, =7 ( = 0.016)). Mice on PN21 had no significant difference between

females (1.01 ± 0.05,𝑛𝑛 =6)𝑃𝑃 and males (1.06 ± 0.06, =7) ( = 0.262).

SFSWAP Expression during𝑛𝑛 the Developmental Ages𝑛𝑛 𝑃𝑃

As age increased from PN0 on to PN21, full-length SFSFWAP (104kDa) relative

expression also increased significantly (Males: PN0: 1.0± 0.05, =6; PN21: 2.21 ± 0.34, =7.

Females: PN0: 1.0± 0.09, =7; PN21: 2.75 ± 0.57, =6) (Males:𝑛𝑛 = 0.009, Females: =𝑛𝑛 0.007)

(Figure 5). Potential isoform𝑛𝑛 of 100kDa SFSWAP a𝑛𝑛lso showed a 𝑃𝑃significant increase in𝑃𝑃 relative

expression as age increases (Males: PN0:1.0 ± 0.07, =6; PN21: 1.52 ± 0.16, =7. Females PN0:

1.0 ± 0.19, =7; PN21: 1.67 ± 0.57, =6) (Males: =𝑛𝑛 0.021, Females: = 0.013)𝑛𝑛 (Figure 5).

𝑛𝑛 𝑛𝑛 𝑃𝑃 𝑃𝑃

Experiment 2: Effects of Testosterone Proprionate on Expression of SFSWAP

Mouse, Tissue, Protein Yield, RNA Yield and Ano-genital Distance

Exp 2.1 Neonatal Treatment:

Measurements of body weight of neonatal treatments of testosterone proprionate pups

that were analyzed for protein did not show any significant sex ( = 0.268), treatment ( =

0.529), or interaction ( = 0.833) differences (Table 2). Tissue weights𝑃𝑃 of these pups indicated𝑃𝑃 no difference in sex ( 𝑃𝑃 = 0.761), but a significant decrease in weight in treated groups ( =

0.003), and a significant𝑃𝑃 decrease in weight in treated males compared to vehicle females𝑃𝑃 ( =

0.049). Protein yield for had no difference in sex ( = 0.657), treatment ( = 0.096), or 𝑃𝑃 interaction ( = 0.185). 𝑃𝑃 𝑃𝑃

Measurements𝑃𝑃 of body weight of neonatal treatments of testosterone proprionate pups that were analyzed for mRNA did not show any significant sex ( = 0.734), treatment ( =

0.179), or interaction ( = 0.734) differences (Table 3). Tissue weights𝑃𝑃 of the 𝑃𝑃

𝑃𝑃 15

cortex/hippocampus of these pups indicated no difference in sex ( = 0.680), no difference in

interaction ( = 0.925), but a significant increase in tissue weight 𝑃𝑃in treated groups ( = 0.024).

RNA Yield showed𝑃𝑃 no significant difference in sex ( = 0.497), treatment ( = 0.156),𝑃𝑃 or interaction ( = 0.156). 𝑃𝑃 𝑃𝑃

Ano-𝑃𝑃genital distance of neonatal treatments of testosterone proprionate pups did show a trend towards a significant difference in sex ( = 0.078), treatment ( = 0.066), and interaction

( = 0.083) (Table 4). 𝑃𝑃 𝑃𝑃

Experiment𝑃𝑃 2.2 Postnatal Treatment:

Measurements of body weight of postnatal treatments of testosterone proprionate pups that were analyzed for protein did not show any significant sex ( = 0.760), treatment ( =

0.838), or interaction ( = 0.812) differences (Table 2). Tissue w𝑃𝑃eights for these mice showed𝑃𝑃 no sex ( = 0.268), treatment𝑃𝑃 ( = 0.529), or interaction ( = 0.833) differences. Protein yield showed𝑃𝑃 no difference in sex𝑃𝑃 ( = 0.284), treatment ( 𝑃𝑃= 0.740), or interaction ( = 0.474).

Measurements of body𝑃𝑃 weight of postnatal treatments𝑃𝑃 of testosterone proprionate𝑃𝑃 pups that were analyzed for mRNA did show a significant difference in sex ( = 0.027), but this was not seen in treatment ( = 0.253), or interaction ( = 0.335) (Table 3). Tissue𝑃𝑃 weights for these

mice showed no sex ( 𝑃𝑃 = 0.789), treatment ( = 0.827),𝑃𝑃 or interaction ( = 0.830) differences.

RNA Yield showed a 𝑃𝑃significant difference in𝑃𝑃 sex ( = 0.048), but no significant𝑃𝑃 difference in treatment ( = 0.323) or interaction ( = 0.928). 𝑃𝑃

Anogenital𝑃𝑃 distance for postnatal𝑃𝑃 treatments of testosterone proprionate pups did show a significant difference in sex ( < 0.001), but did not show this difference in treatment ( =

0.435) or interaction ( = 0.758)𝑃𝑃 (Table 4). 𝑃𝑃

Sfswap mRNA Expression𝑃𝑃 for Neonatal Treatments of Testosterone Proprionate

Sfswap had no significant sex difference (p=0.997), treatment difference (p=0.253), or interaction difference (p=0.759). Sfswap in vehicle and TP0 had relatively constent expression in both males and females (Male Vehicle: 1.11 ± 0.37, =10; Male TP0: 1.22 ± 0.47, =10;

Female Vehicle: 1.07 ± 0.28, =10; Female TP0: 1.26𝑛𝑛 ± 0.25, = 10) (Figure 6a).𝑛𝑛

SFSWAP Expression for Neonatal𝑛𝑛 Treatments of Testosterone Proprionate𝑛𝑛

SFSWAP expression for TP0 mice compared to vehicle treated mice indicated there was no differences in sex ( =0.408), treatment ( =0.203), or interaction ( =0.108) (Vehicle PN0 males: =4, Vehicle𝑃𝑃 PN0 females: = 2, Testosterone𝑃𝑃 Proprionate treated𝑃𝑃 PN0 males: =3,

Testosterone𝑛𝑛 Proprionate treated females:𝑛𝑛 =5) (Figure 6b). 𝑛𝑛

Sfswap mRNA expression for Postnatal Treatments𝑛𝑛 of Testosterone Proprionate

Sfswap expression for TP23 indicated there was no differences in sex ( =0.253), treatment

( =0.144), or interaction ( =0.437) (Vehicle PN23 males: =12, Vehicle PN23𝑃𝑃 females: =12,

Testosterone𝑃𝑃 Proprionate treated𝑃𝑃 PN23 males: =12, Testosterone𝑛𝑛 Proprionate treated PN23𝑛𝑛 females: =12) (Figure 7a). 𝑛𝑛

SFSWAP𝑛𝑛 Expression for Postnatal Treatments of Testosterone Proprionate

SFSWAP expression for TP23 indicated that there was no difference in sex (p=0.588), treatment ( =0.066), but a significant difference in interaction ( =0.032) (Figure 7b). Female

Sfswap had𝑃𝑃 a significant decrease in expression when treated (1.00𝑃𝑃 ± 0.08 for V, =6 to 0.66 ±

0.07 for TP, =6). Males had no significant difference when treated (0.86 ± 0.09 𝑛𝑛for V, =6 to

0.89 ± 0.07 for𝑛𝑛 TP, =6). 𝑛𝑛

𝑛𝑛

Experiment 3: Effects of Testicular Feminization Mutation on Expression of SFSWAP

Mouse, Tissue, Protein Yield and Ano-genital Distance

Measurements of mouse body weight ( =0.158) and tissue weight ( =0.258) showed no difference in male, female, and Tfm mice (Table𝑃𝑃 2). Anogenital distance showed𝑃𝑃 males had significantly larger distances ( =0.039) than females or Tfm mice (Table 4). Protein yield showed no significant difference𝑃𝑃 between males, females, or Tfm mice ( =0.374).

SFSWAP Expression 𝑃𝑃

Tfm mice, lacking functional AR, showed SFSWAP levels similar to wildtype females

(Tfm: 0.86± 0.19, =5; WT Females: 1.00± 0.16, =6; WT males: 0.64± 0.16, =4) (Figure 8).

Tfm mice exhibit a𝑛𝑛 trend in higher SFSWAP levels𝑛𝑛 than wild type males ( = 0.182).𝑛𝑛 These data are preliminary with a low number. 𝑃𝑃

𝑛𝑛

CHAPTER 4

DISCUSSION

Sexually Dimorphic SFSWAP Expression during Development

Significantly more female SFSWAP protein (52% greater in female PN7 mice) than male protein was observed. This female bias continues on PN14 (25% greater in female PN14 mice) and PN21 (28% greater in female PN21 mice).This variation in amount of SFSWAP in the cortex/hippocampus can dramatically alter its downstream products in a sex biased manner.

Alternative splicing of these downstream factors can further increase the abundance of these sex differences (Chen and Manley 2009). Alternative splicing is a fundamental process where cells can expand the diversity of their transcripts. Males and/or females may result in varying functions of neural systems and behaviors because of these processes. Regulation of this process, which is the function of splicing factors such as Sfswap, is critical for modulating protein-protein interactions, transcription networks, and multiple aspects of neuronal development. (Raj and

Blencowe 2015). One possible reason that Sfswap is sexually dimorphic during the developmental ages is due to the role of neuroanatomical maturation during this time period. The cortex develops throughout childhood and adolescence in humans as cortical surface area increases with age and is larger in females (Sussman et al., 2016). Additionally in rats, the male hippocampus differs in size in females and is due to higher rates of cell genesis during development compared to females (Bowers et al., 2010). Since Sfswap is found ubiquitously throughout the brain, these anatomical differences in male and female brain structures can act on and establish the observed gender differences in SFSWAP expression. Therefore, the sex difference observed in SFSWAP on PN7, PN14, and PN21 may be established due to the formation of this sex specific development of the cortex/hippocampus. The alternative may also

be true; that sexually dimorphic SFSWAP induces the anatomical sex differences in tissue of

these brain regions. SFSWAP regulates splicing of specific gene transcripts in the

cortex/hippocampus which can have significant effects on the synthesis of protein, cells and

tissues. Its specific splicing regulation may activate the development of the anatomical difference

of the brain in males and females. This, coupled with the ability of Sfswap to regulate alternative

splicing of even itself, may distinguish Sfswap as a key gene causing many downstream sex differences in the male and female cortex/hippocampus.

The specific female bias of SFSWAP has several possible consequences. One possible consequence of female biased SFSWAP expression is the direct downstream genes of Sfswap. As previously stated, Sfswap enhances splicing of CD45 genes and represses splicing of MAPT/Tau exon 10. CD45 is critical in regulation of several kinases in T cells and B cells and overall immune function (Donovan and Koretzky 1993). It is also important in neuronal maintenance along with removal of neurotoxic Aβ oligomer, which is key in preventing neuronal loss in

Alzheimer’s disease, a significant neurological disease higher in females than males (Zhu et al.,

2011);(Vina and Lloret 2010). SFSWAP, therefore, not only regulates splicing of key genes in the brain but may also regulate neurological disorders associated with those diseases. Further studies are still needed to elucidate the direct connections between Sfswap and these neurological disorders. Understanding the nature of these sex differences and the influence Sfswap has on this system is critical in further characterizing these brain regions and developing treatments for these associated diseases.

Another possible consequence of a female bias in SFSWAP expression, is the possibility of differences in sex on regulation of maintenance and formation of inner ear organs. Sfswap is a critical regulator of growth and patterning of inner ear sensory organs and is key in maintaining

cells in these regions of the ear (Moayedi et al., 2014). However, my study looks specifically at

the expression of SFSWAP in the cortex/hippocampus. The interactions between the vestibular

inner ear and the hippocampus are necessary in the development of spatial memory during

learning (Zheng et al., 2003). The cortex interacts with the vestibular apparatus of the inner ear

to influence multisensory processing and elementary perceptions (Bottini et al., 2013). This interaction between the cortex/hippocampus and the inner ear suggests a possible role that

SFSWAP may be linked to sex differences in the inner ear. A recent publication that examined

C57BL/6J mice and uncovered changes in neuron numbers of the cochlear nucleus between the sexes (Willott 2009). This sex difference in the cochlear nucleus may be a product of the sex difference observed in SFSWAP. Surprisingly, increasing evidence also indicates that sex hormones can influence the development and interactions of sensory cells and neurons of the auditory system (Canlon and Frisina 2009). This may be critical in understanding sex differences not only in the development of the cortex/hippocampus but also in the inner ear, making Sfswap critical in both.

It is interesting to note, however, that the female bias in SFSWAP does not begin until PN7.

This may be due to effects of testosterone. As previously stated, males experience two peaks of testosterone during birth, once at E16 and once around birth (PN0). With the possibility that T may regulate SFSWAP during development, this age dependent change in increased female expression of SFSWAP may be due to slow activation of T. Testosterone actions may vary considerably and the activation of its effects have a specific time dependent course (Foradori et al., 2008; Saad et al., 2011). When T acts via androgen receptor (AR) that binds specific DNA response elements in target gene promoters, transcription is activated and can therefore drive protein synthesis (Tsai and O'Malley 1994). In recent years, a new non-genomic pathway that T

may alternatively act is by directly activating membrane receptors and therefore activating

secondary messengers inducing their cascades (Michels and Hoppe 2008). Along with this data,

it has been suggested that prenatal T might alter gene expression around the time of birth due to

transcriptional regulation of brain specific genes by histone modifications (Tsai et al., 2009).

Whichever pathway testosterone takes, its effects would not be observed in PN0 SFSWAP expression. This is because the testosterone surge begins a few hours after birth, already after these mice were dissected for analysis. The effects would then be observed on PN7 and so forth, which is the case. With increasing evidence suggests that AR may play a strong role in the brain development and neuronal protection in regions affected by Alzheimer’s disease (Kerr et al.,

1995; Simerly et al., 1990; Tohgi et al., 1995), this links testosterone via AR to repress the protein synthesis of SFSWAP in males which is not observed in females.

Potential Isoforms of SFSWAP

Based off of observational analysis, SFSWAP has multiple significant bands shown during

immunoblotting. Here, I identify one of these bands (100kDa) closest to the more identified,

literature based molecular weight of 104kDa. This band was analyzed for sex differences, which

were observed on PN7 and PN14. This differed to SFSWAP 104kDa by not expressing a female

bias at PN21. Although further analysis is needed to confirm that this is an isoform using cluster

analysis of MALDI-MS (Alm et al., 2006), one presumptive method of their origin may be due

to the self-splicing nature of Sfswap. Alternative splicing of SFSWAP would increase the protein

isoform diversity dramatically and could result in these smaller variants of the full length protein.

Regulation of these potential isoforms and the full length protein are key in further understanding

their downstream effects on sex differences in the cortex/hippocampus.

Difference in mRNA and Protein Expression of Sfswap as age increases

In the expression of Sfswap, there seems to be a dramatic variation in the measured mRNA levels and the protein levels. Sfswap mRNA expression shows only a sex difference on

PN7 but this is not observed on PN0, PN14, and PN21. SFSWAP protein, on the other hand, expresses a sex difference on PN7, PN14, and PN21, and not on PN0. In addition to this, the relative abundance of mRNA decreases with age and the relative abundance of protein increases with age. The observed discrepancy between mRNA and protein is becoming more understood.

In recent years, published studies on the correlation of mRNA and corresponding protein levels in complex samples are not very abundant. (Gygi et al., 1999; Maier et al., 2009; Washburn et al., 2003). Transciption and translation are far more complex than simple and linear relationships. A variety of events may therefore result in this uncoupling of transcription and translation in Sfswap mRNA and protein. For instance, features of regulatory proteins and sRNAs can act as translational modulators changing trancriptiona and translation rates (Golding et al., 2005; Gottesman 2004). Additionally variation in ribosome location and attachment may play a role (Arava et al., 2003) and even mRNA distribution and sequestration also influences translation rates. Regulation of its own transcript of Sfswap, by splicing out its own first two introns (Denhez and Lafyatis 1994), may also result in this difference in mRNA and protein levels.

Effects of Testosterone Proprionate on Expression of SFSWAP

Only when treated postnatally with testosterone proprionate did SFSWAP females experience a decrease in expression. This was interestingly not observed in males. This female

SFSWAP selective sensitivity to testosterone may be due to a variety of reasons. This difference may be a consequence of additional modulators of gene expression that affect the way T would

23 bind and interact with the genome. Estrogen receptors (ERs) are known to indirectly interact with the receptor for testosterone, AR, acting on and developing the brain (Morris et al., 2004).

Expression of Cytochrome P450 19A1, the aromatase responsible for the enzymatic conversion of T to estradiol (Lephart 1996), is more highly expressed in the hypothalamus of testosterone treated female juncas rather than control females (Peterson et al., 2013). Higher levels of circulating testosterone would therefore result in higher levels of estradiol in females. The effects of estradiol are known to mediate many of the well-known developmental and regulational effects of T (Forlano et al., 2006). This suggests that perhaps the regulation of SFSWAP by T via

AR, may actually be mediated through estradiol. Furthermore, this indicates that the sexes may process or interpret hormonal signals differently, which is consistent with our finding that T- treatment affects SFSWAP differently in each sex.

T may also act directly to modify the genome of Sfswap. It has been shown that T regulates a significant increase in H3K9/14Ac, a specific histone found in the cortex/hippocampus (Tsai et al., 2009). Histone modifications have been demonstrated to regulate transcription of brain- specific genes therefore changing neural function and behavior in mice and rats (Forger et al.,

2004; McCarthy et al., 2009; Murray et al., 2009). This suggests that during development, T regulation of Sfswap may result in the key neural development between the sexes.

Effects of Testicular Feminization Mutation on Expression of SFSWAP

In this study, mice were bred with testicular feminization mutation (Tfm), a naturally occurring point mutation of the gene encoding the androgen receptor. These mice are unable to respond to physiological levels of androgens (Langley et al., 1998; Yarbrough et al., 1990).

Androgen insensitivity alters the reproductive ability of these mice developing feminized genitalia and lowered responses of masculine sex behavior (Hamson et al., 2009; Olsen and

Whalen 1981). Taken together, this mutation allows scientists to not only understand the role of

androgen receptors in the brain, but also the effects it has on other mechanisms. In my case, my

interest lies in the developmental regulation that testosterone via AR has on SFSWAP.

Administration of testosterone can offer favorable data towards understanding the function of

AR, but it can also produce a variety of effects that rely on numerous pathways (aromatization to

estradiol, secondary messenger cascades, etc.) In the experiments presented here, I directly

illustrate the effect of AR has on SFSWAP expression. SFSWAP expression in male Tfm mice

had similar levels as wild type female expression. Both wild type females and male Tfm mice

were found to be trending toward significantly higher expression than wildtype male SFSWAP.

My results implicate androgen sensitive SFSWAP cells are regulated by AR, resulting in their

decrease in expression.

Hypothetic Working Model on the Regulation of SFSWAP

Testosterone is a key sex hormone altering the mechanisms that give rise to sex differences

in structure and behavior of the brain. Testosterone is critical in the development of the nervous

system, promoting or inhibiting cell death and regulating formation of synapses (Estrada et al.,

2006; Morris et al., 2004). Therefore, the two surges of testosterone released prenatally and around birth in mice, may be critical in forming sex differences in the cortex/hippocampus. The particular cells and specific genes whereby testosterone acts to induce these effects are far understudied at the moment.

Increasing evidence suggests that sex differences are established in the brain due to differential apoptosis in male and female brain regions. Neonatal male and female mice initially have the same number of cells in certain brain regions such as the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV). (Zhen, et

al., 2013) However, these two brain regions quickly establish staggering sex differences due to

the sex hormone estrogen (Davis et al., 1996; Waters and Simerly 2009), causing apoptosis in

male AVPV and reducing cell death in the SDN-POA. It is important to note that the sex difference is induced by differential apoptosis between the sexes rather than the more likely considered notion of cell genesis.

I propose here that SFSWAP is regulated by the perinatal rise in testosterone (Figure 9). This rise in testosterone may be acting through a similar pathway whereby sex differences are induced in the SDN-POA and the AVPV. Two subpopulations of SFSWAP-expressing cells speculated

to exist in neonatal cortex/hippocampus; one coexpresses AR (+) and the other not. Both types of

SFSWAP cells proliferate as age advances. The perinatal rise in T activates AR to cause cell

death specifically in AR-positive SFSWAP cells in male mice while the same subpopulation is

not affected and continues to proliferate in females due to the lack of T. As the result of such

effect of AR, male mice not only display the lower number of SFSWP-expressing cells in the

cortex/hippocampus than females, but also lose their responsiveness to T due to T-induced

selective apoptosis. Overall, dimorphic expression of SFSWAP might result in sex-biased

splicing regulation of its target genes, which controls differential development and function of

the cortex/hippocampus between the sexes and possibly introduces gender biases in many

neurological disorders and mental illness.

APPENDICES

APPENDIX A

TABLES

Table 1. List of Oligonucleotide Primers used for RT-PCR and RT-qPCR.

Product Annealing Gene RefSeq Forward Primer Reverse Primer Size Temperature Symbol Accession (5'-3') (5'-3') (bp) (°C) Efficiency

CCAGAGGCA 87% NM_007 CCAGATCATGTT Actb TACAGGGAC 78 59 393.3 TGAGACCTTCAA AGC

CAGGAGTCC 57% NM_009 ATGAGGTCGGGT Rpl13a GTTGGTCTTG 179 62 438.5 GGAAGTACC AG

CAGGAAGGG 85% NM_172 GAGTACACGGCA Sfswap TGCTGTAGTA 84 60 276 GACTCAACT AGT

Table 2. Mouse weights, tissue weights, and protein yield used for protein analysis

Sample Group N Body Weight (g) Tissue Weight (mg) Protein Yield (µg) Experiment 1- PN0 Females 24 1.27 ± 0.02 28.1 ± 2.36 31.19 ± 2.32 Males 28 1.23 ± 0.09 30.94 ± 2.19 31.15 ± 3.87 PN7 Females 24 4.27 ± 0.17 85.43 ± 3.62 66.26 ± 5.86 Males 28 3.77 ± 0.12 93.13 ± 9.55 66.42 ± 4.72 PN14 Females 24 7.17 ± 0.25 114.39 ± 13.82 51.79 ± 4.43 Males 28 7.94 ± 0.11 115.66 ± 9.09 46.68 ± 3.65 PN21 Females 24 7.82 ± 0.55 123.18 ± 7.47 28.39 ± 1.25 Males 28 8.53 ± 0.31 119.66 ± 3.94 30.32 ± 1.59 Experiment 2- PN0 Vehicle Females 2 1.55 ± 0.05 69.95 ± 0.75 21.84 ± 0.94 Vehicle Males 4 1.28 ± 0.03 74.50 ± 2.01 23.95 ± 0.21 TP Females 5 1.38 ± 0.03 65.88 ± 2.41 20.93 ± 2.29 TP Males 3 1.17 ± 0.09 59.87 ± 2.11 16.84 ± 2.60 PN21 Vehicle Females 6 8.85 ± 0.47 162.4 ± 12.21 55.38 ± 3.12 Vehicle Males 6 8.88 ± 0.36 148.83 ± 6.81 49.59 ± 3.30 TP Females 6 8.83 ± 0.32 153.83 ± 4.03 52.01 ± 1.42 TP Males 6 9.1 ± 0.44 144.55 ± 18.81 50.84 ± 4.12 Experiment 3- Females 7 10.46 ± 0.91 48.87 ± 11.41 55.23 ± 2.24 Males 4 8.1 ± 0.92 103.75 ± 42.83 46.48 ± 4.20 Tfm 5 9.31 ± 0.31 88.67 ± 22.78 53.06 ± 5.50 Data are shown as mean±SD

Table 3. Mouse weights, tissue weights and RNA yield used for mRNA analysis Extracted RNA Sample Group N Body Weight (g) Tissue Weight (mg) Yield (µg) Experiment 1- PN0 Females 13 1.24 ± 0.03 24.54 ± 7.93 46.1 ± 4.28 Males 10 1.24 ± 0.15 21.04 ± 3.89 40.71 ± 3.43 PN7 Females 9 3.78 ± 0.49 75.87 ± 22.54 105.72 ± 10.64 Males 8 3.49 ± 0.55 79.29 ± 10.33 105.66 ± 8.12 PN14 Females 8 7.64 ± 1.30 100.61 ± 22.08 128.23 ± 13.16 Males 8 7.18 ± 1.07 97.43 ± 17.41 138.94 ± 19.73 PN21 Females 7 8.73 ± 1.69 109.59 ± 23.45 115.11 ± 19.82 Males 9 8.90 ± 0.67 119.26 ± 19.46 127.04 ± 16.87 Experiment 2- PN0 Vehicle Females 10 1.29 ± 0.04 18.99 ± 1.53 48.20 ± 3.93 Vehicle Males 10 1.29 ± 0.03 18.43 ± 2.18 39.80 ± 2.56 TP Females 10 1.24 ± 0.02 23.27 ± 1.24 36.80 ± 2.78 TP Males 10 1.26 ± 0.02 22.38 ± 1.87 29.80 ± 5.69 PN21 Vehicle Females 11 7.76 ± 0.48 99.90 ± 8.62 206.00 ± 13.81 Vehicle Males 11 8.35 ± 0.62 100.34 ± 10.68 184.55 ± 11.29 TP Females 11 7.85 ± 0.22 96.39 ± 7.03 196.00 ± 10.53 TP Males 11 9.29 ± .34 100.3 ± 4.60 172.55 ± 7.33 Data are shown as mean±SD

Table 4. Anogenital Distance (AGD)

Sample Group N AGD (mm) Experiment 1- PN0 Females 37 - Males 38 - PN7 Females 35 2.55 ± 0.14 Males 36 3.43 ± 0.22 PN14 Females 32 3.64 ± 0.18 Males 36 5.50 ± 0.41 PN21 Females 31 3.75 ± 0.31 Males 37 6.00 ± 0.50 Experiment 2- PN0 Vehicle Females 10 1.86 ± 0.07 Vehicle Males 10 1.87 ± 0.03 TP Females 10 1.91 ± 0.06 TP Males 10 2.21 ± 0.12 PN21 Vehicle Females 11 4.53 ± 0.43 Vehicle Males 11 6.64 ± 0.56 TP Females 11 4.11 ± 0.23 TP Males 11 6.46 ± 0.19 Experiment 3- Females 7 3.49 ± 0.30 Males 4 5.51 ± 0.97 Tfm 5 3.26 ± 0.33 Data are shown as mean of Outer AGD and Inner AGD ±SD - indicates AGD was not measured at time of collection

APPENDIX B

FIGURES

* 1.5 F M 1.0 *

0.5 mRNA levels (vs. 0-F)

13 10 9 8 8 8 7 9 0.0 Sfswap 0 7 14 21 Age (days after birth)

Figure 1. Expression of mRNA Sfswap at the developmental ages. Expression shows no sex difference but a significant age decrease in Sfswap from PN0 on PN14 and PN21 * indicates a significant difference, p < 0.05. Numbers indicate sample size.

Figure 2. Representative immunoblotting of SFSWAP protein in male (M) and female (F) on

age. Post natal day of birth (PN7) is shown. Full length SFSWAP (104kDA) detected in the mouse cortex/ hippocampus. Various alternative bands are shown as well indicating possible alternative splice variants such as at 100kDa.

1.5 ) 1.5 PN7 PN0 F

. s v (

1.0 4 1.0 0 1

0.5 A 0.5 * W S

7 8 F 7 8 S SFSWAP 104 (vs. F) 0.0 0.0 F M F M Sex Sex ) 1.5 PN14 ) 1.5 PN21 F F

. . s s v v ( (

4 4 1.0 1.0 0 0 1 1

P P * * A A 0.5 0.5 W W S S F F 7 8 7 8 S S 0.0 0.0 F M F M Sex Sex

Figure 3. Expression of SFSWAP 104kDa protein in the cortex/hippocampus of male and female mouse pups on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) after birth. Mean optical densities (±SEM) of SFSWAP protein were normalized to levels of β-actin and expressed as the fold of females with the same ages (as 1 fold). * indicates significant difference, p < 0.05.

Numbers indicate sample size.

) 1.5 PN0 F PN7 1.5 . s v (

1.0 0 1.0 0 1

P * 0.5 A 0.5 W S

F 7 8 7 8 S SFSWAP(vs.100 F) 0.0 0.0 F M F M Sex Sex

) 1.5 ) 1.5

F PN14 F PN21

. . s s v v ( (

0 1.0 0 1.0 0 0 1 1 * P P

A 0.5 A 0.5 W W S S

F F 8

S 7 8 S 7 0.0 0.0 F M F M Sex Sex

Figure 4. Expression of SFSWAP 100kDa protein in the cortex/hippocampus of male and female mouse pups on the day of birth (PN0), and 7 (PN7), 14 (PN14), and 21 (PN21) after birth. Mean optical densities (±SEM) of SFSWAP protein were normalized to levels of β-actin and expressed as the fold of females with the same ages (as 1 fold). * indicates significant difference, p < 0.05.

Numbers indicate sample size.

Female Female 4 ) 4 0

* s

3 v 3 (

0 * 0

2 1 2

p a

1 w 1 s f S Sfswap 104 (vs. 0) 8 7 8 7 0 0 0 21 0 21 Age (days after birth) Age (days after birth)

Male Male ) 4 4 0 ) 0 . s . v v s 3 3 * ( 4 ( 0 0 * 2 2 1 p p 1 a a

w 1 1 s w s f f S S 8 7 8 7 0 0 0 21 0 21 Age (days after birth) Age (days after birth)

Figure 5. Expression of SFSWAP protein in the cortex/hippocampus comparing age in mouse

pups on the day of birth (PN0) and 21 (PN21) days after birth. Mean optical densities (±SEM) of

SFSWAP protein were normalized to levels of β-actin and expressed as the fold of females with the same ages (as 1 fold). * indicates significant difference, p < 0.05. Numbers indicate sample size.

[a]

2.0 F M 1.5

1.0

mRNA levels (vs. V-F) 0.5

10 10 10 10 0.0 Sfswap V TP Treatment

[b]

2.0 F M 1.5

1.0

0.5 Sfswap 104 (vs. V-F) 2 4 5 3 0.0 V TP Treatment

Figure 6. Relative expression levels of Sfswap in the prenatal cortex/hippocampus of male (M) and female (F) mice treated with vehicle (V) and testosterone proprionate (TP). (a) Sfswap mRNA levels were quantified relative to average in vehicle females. (b) SFSWAP protein levels represented as mean optical densities (±SEM) that were normalized to levels of β-actin and expressed as the fold of V-F (as 1 fold). Numbers indicate sample size.

[a]

2.5 F 2.0 M

1.5

1.0

mRNA levels (vs. V-F) 0.5

11 11 11 11 0.0 Sfswap V TP Treatment

[b]

1.5 * F M 1.0

0.5

SFSWAP 104 (vs. V-F) 6 6 6 6 0.0 V TP Treatment

Figure 7. Relative expression levels of Sfswap in the cortex/hippocampus of postnatal male (M) and female (F) mice treated with vehicle (V) and testosterone proprionate (TP). (a) Sfswap mRNA levels were quantified as relative to average in vehicle females. (b) SFSWAP protein levels represented as mean optical densities (±SEM) that were normalized to levels of β-actin and expressed as the fold of V-F (as 1 fold). * indicates significant difference, p < 0.05. Number indicate sample size.

1.5

1.0 #

104 levels (vs. V-F) 0.5

7 4 5 0.0 SFSWAP F M Tfm

Figure 8. Relative expression levels of SFSWAP in the cortex/ hippocampus of testicular

feminization mutation (Tfm) mice as well as wild-type male (M) and female (F) littermates at

PN21. The SFSWAP level is normalized to β-actin as compared to F (set as 1 fold). # indicates similar trend in sex difference. Results are preliminary with a low N number. Numbers indicate sample size.

Figure 9. Hypothetic working model that illustrates the development of sexually dimorphic

SFSFWAP expression in the developing mouse cortex/hippocampus. Black circles indicate cell.

+ indicates cells coexpressing AR. Arrows indicate progression from PN0 to PN21 accompanied with cell proliferation.

References

Alm, R., Johansson, P., Hjerno, K., Emanuelsson, C., Ringner, M., Hakkinen, J., 2006. Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra. J Proteome Res. 5, 785-792.

Antunes-Martins, A., Mizuno, K., Irvine, E.E., Lepicard, E.M., Giese, K.P., 2007. Sex-dependent up-regulation of two splicing factors, psf and Srp20, during hippocampal memory formation. Learn Mem. 14, 693-702.

Arava, Y., Wang, Y., Storey, J.D., Liu, C.L., Brown, P.O., Herschlag, D., 2003. Genome-wide analysis of mRNA translation profiles in saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 100, 3889-3894.

Baron-Cohen, S., Lombardo, M.V., Auyeung, B., Ashwin, E., Chakrabarti, B., Knickmeyer, R., 2011. Why are autism spectrum conditions more prevalent in males? PLoS Biol. 9, e1001081.

Baron-Cohen, S., Auyeung, B., Ashwin, E., Knickmeyer, R., 2009. Fetal testosterone and autistic traits: A response to three fascinating commentaries. Br J Psychol. 100, 39-47.

Bonthuis, P.J., Cox, K.H., Searcy, B.T., Kumar, P., Tobet, S., Rissman, E.F., 2010. Of mice and rats: Key species variations in the sexual differentiation of brain and behavior. Front Neuroendocrinol. 31, 341-358.

Bottini, G., Gandola, M., Sedda, A., Ferre, E.R., 2013. Caloric vestibular stimulation: Interaction between somatosensory system and vestibular apparatus. Front Integr Neurosci. 7, 66.

Bowers, J.M., Waddell, J., McCarthy, M.M., 2010. A developmental sex difference in hippocampal neurogenesis is mediated by endogenous oestradiol. Biol Sex Differ. 1, 8-6410-1-8.

Breedlove, S.M., Hampson, E., 2002. Sexual differentiation of the brain and behavior. In Behavioral Endocrinology. J.B. Becker, S.M. Breedlove, D. Crews, M.M. McCarthy, eds. MIT Press, pp. 75.

Canlon, B., Frisina, R.D., 2009. Sex hormones and hearing: A pioneering area of enquiry. Hear Res. 252, 1-2.

Carper, R.A., Moses, P., Tigue, Z.D., Courchesne, E., 2002. Cerebral lobes in autism: Early hyperplasia and abnormal age effects. Neuroimage. 16, 1038-1051.

Chen, M., Manley, J.L., 2009. Mechanisms of alternative splicing regulation: Insights from molecular and genomics approaches. Nat Rev Mol Cell Biol. 10, 741-754.

Corbier, P., Edwards, D.A., Roffi, J., 1992. The neonatal testosterone surge: A comparative study. Arch Int Physiol Biochim Biophys. 100, 127-131.

Cuadrado, A., Bernal, J., Munoz, A., 1999. Identification of the mammalian homolog of the splicing regulator suppressor-of-white-apricot as a thyroid hormone regulated gene. Brain Res Mol Brain Res. 71, 332-340.

Davis, E.C., Popper, P., Gorski, R.A., 1996. The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain Res. 734, 10-18.

Demir, E., Dickson, B.J., 2005. Fruitless splicing specifies male courtship behavior in drosophila. Cell. 121, 785-794.

Deng, Z., Sobell, J.L., Knowles, J.A., 2010. Epigenetic alterations in schizophrenia. FOCUS: The Journal of Lifelong Learning in Psychiatry. 8, 358-365.

Denhez, F., Lafyatis, R., 1994. Conservation of regulated alternative splicing and identification of functional domains in vertebrate homologs to the drosophila splicing regulator, suppressor-of- white-apricot. J Biol Chem. 269, 16170-16179.

Dickson, P.W., Aldred, A.R., Menting, J.G., Marley, P.D., Sawyer, W.H., Schreiber, G., 1987. Thyroxine transport in choroid plexus. J Biol Chem. 262, 13907-13915.

Dillman, A.A., Hauser, D.N., Gibbs, J.R., Nalls, M.A., McCoy, M.K., Rudenko, I.N., Galter, D., Cookson, M.R., 2013. mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex. Nat Neurosci. 16, 499-506.

Donovan, J.A., Koretzky, G.A., 1993. CD45 and the immune response. J Am Soc Nephrol. 4, 976-985.

Doyle-Thomas, K.A., Duerden, E.G., Taylor, M.J., Lerch, J.P., Soorya, L.V., Wang, A.T., Fan, J., Hollander, E., Anagnostou, E., 2013. Effects of age and symptomatology on cortical thickness in autism spectrum disorders. Res Autism Spectr Disord. 7, 141-150.

Dredge, B.K., Polydorides, A.D., Darnell, R.B., 2001. The splice of life: Alternative splicing and neurological disease. Nat Rev Neurosci. 2, 43-50.

Dussault, J.H., Ruel, J., 1987. Thyroid hormones and brain development. Annu Rev Physiol. 49, 321-334.

Estrada, M., Varshney, A., Ehrlich, B.E., 2006. Elevated testosterone induces apoptosis in neuronal cells. J Biol Chem. 281, 25492-25501.

Foradori, C.D., Weiser, M.J., Handa, R.J., 2008. Non-genomic actions of androgens. Front Neuroendocrinol. 29, 169-181.

Forger, N.G., 2009. Control of cell number in the sexually dimorphic brain and spinal cord. J Neuroendocrinol. 21, 393-399.

Forger, N.G., Rosen, G.J., Waters, E.M., Jacob, D., Simerly, R.B., de Vries, G.J., 2004. Deletion of bax eliminates sex differences in the mouse forebrain. Proc Natl Acad Sci U S A. 101, 13666- 13671.

Forlano, P.M., Schlinger, B.A., Bass, A.H., 2006. Brain aromatase: New lessons from non- mammalian model systems. Front Neuroendocrinol. 27, 247-274.

Fraboulet, S., Boudouresque, F., Delfino, C., Fina, F., Oliver, C., Ouafik, L., 1996. Effect of thyroid hormones on peptidylglycine alpha-amidating monooxygenase gene expression in anterior pituitary gland: Transcriptional studies and messenger ribonucleic acid stability. Endocrinology. 137, 5493-5501.

Frick, K.M., Gresack, J.E., Dec 2003. Sex differences in the behavioral response to spatial and object novelty in adult C57BL/6 mice. Vol 117, 1283-1291.

Golding, I., Paulsson, J., Zawilski, S.M., Cox, E.C., 2005. Real-time kinetics of gene activity in individual bacteria. Cell. 123, 1025-1036.

Gottesman, S., 2004. The small RNA regulators of escherichia coli: Roles and mechanisms*. Annu Rev Microbiol. 58, 303-328.

Greer, J.M., McCombe, P.A., 2011. Role of gender in multiple sclerosis: Clinical effects and potential molecular mechanisms. J Neuroimmunol. 234, 7-18.

Gygi, S.P., Rochon, Y., Franza, B.R., Aebersold, R., 1999. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 19, 1720-1730.

Hamson, D.K., Csupity, A.S., Ali, F.M., Watson, N.V., 2009. Partner preference and mount latency are masculinized in androgen insensitive rats. Physiol Behav. 98, 25-30.

Hines, M., 2006. Prenatal testosterone and gender-related behaviour. Eur J Endocrinol. 155 Suppl 1, S115-21.

Ivanova, T., Beyer, C., 2000. Ontogenetic expression and sex differences of aromatase and estrogen receptor-α/β mRNA in the mouse hippocampus. Cell Tissue Res. 300, 231-237.

Jazin, E., Cahill, L., 2010. Sex differences in molecular neuroscience: From fruit flies to humans. Nature Reviews Neuroscience. 11, 9-17.

Kar, A., Kuo, D., He, R., Zhou, J., Wu, J.Y., 2005. Tau alternative splicing and frontotemporal dementia. Alzheimer Dis Assoc Disord. 19 Suppl 1, S29-36.

Kerr, J.E., Allore, R.J., Beck, S.G., Handa, R.J., 1995. Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology. 136, 3213-3221.

Kopsida, E., Stergiakouli, E., Lynn, P.M., Wilkinson, L.S., Davies, W., 2009. The role of the Y chromosome in brain function. Open Neuroendocrinol J. 2, 20-30.

Langley, E., Kemppainen, J.A., Wilson, E.M., 1998. Intermolecular NH2-/carboxyl-terminal interactions in androgen receptor dimerization revealed by mutations that cause androgen insensitivity. J Biol Chem. 273, 92-101.

Lephart, E.D., 1996. A review of brain aromatase cytochrome P450. Brain Res Brain Res Rev. 22, 1-26.

Maier, T., Güell, M., Serrano, L., 2009. Correlation of mRNA and protein in complex biological samples. FEBS Lett. 583, 3966-3973.

Markham, J., Jurgens, H., Auger, C., De Vries, G., Arnold, A., Juraska, J., 2003. Sex differences in mouse cortical thickness are independent of the complement of sex chromosomes. Neuroscience. 116, 71-75.

McCarthy, M.M., Auger, A.P., Bale, T.L., De Vries, G.J., Dunn, G.A., Forger, N.G., Murray, E.K., Nugent, B.M., Schwarz, J.M., Wilson, M.E., 2009. The epigenetics of sex differences in the brain. The Journal of Neuroscience. 29, 12815-12823.

McCarthy, M.M., Arnold, A.P., 2011. Reframing sexual differentiation of the brain. Nat Neurosci. 14, 677-683.

McCarthy, M.M., Ball, G.F., 2008. The neuroendocrine control of sex specific behavior in vertebrates: Lessons from mammals and birds. Curr Top Dev Biol. 83, 213-248.

Merkley, C.M., Jian, C., Mosa, A., Tan, Y.F., Wojtowicz, J.M., 2014. Homeostatic regulation of adult hippocampal neurogenesis in aging rats: Long-term effects of early exercise. Front Neurosci. 8, 174.

Michels, G., Hoppe, U.C., 2008. Rapid actions of androgens. Front Neuroendocrinol. 29, 182- 198.

Moayedi, Y., Basch, M.L., Pacheco, N.L., Gao, S.S., Wang, R., Harrison, W., Xiao, N., Oghalai, J.S., Overbeek, P.A., Mardon, G., Groves, A.K., 2014. The candidate splicing factor sfswap regulates growth and patterning of inner ear sensory organs. PLoS Genet. 10, e1004055.

Morris, J.A., Jordan, C.L., Breedlove, S.M., 2004. Sexual differentiation of the vertebrate nervous system. Nat Neurosci. 7, 1034-1039.

Motelica-Heino, I., Castanier, M., Corbier, P., Edwards, D.A., Roffi, J., 1988. Testosterone levels in plasma and testes of neonatal mice. J Steroid Biochem. 31, 283-286.

Mu, Y., Gage, F.H., 2011. Adult hippocampal neurogenesis and its role in alzheimer's disease. Mol Neurodegener. 6, 85-1326-6-85.

Murray, E.K., Hien, A., de Vries, G.J., Forger, N.G., 2009. Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis. Endocrinology. 150, 4241-4247.

Mylvaganam, R., Ahn, Y.S., Sprinz, P.G., Garcia, R.O., Harrington, W.J., 1989. Sex difference in the CD4 + CD45R+ T lymphocytes in normal individuals and its selective decrease in women with idiopathic thrombocytopenic purpura. Clin Immunol Immunopathol. 52, 473-485.

Ngun, T.C., Ghahramani, N., Sanchez, F.J., Bocklandt, S., Vilain, E., 2011. The genetics of sex differences in brain and behavior. Front Neuroendocrinol. 32, 227-246.

Olsen, K.L., Whalen, R.E., 1981. Hormonal control of the development of sexual behavior in androgen-insensitive (tfm) rats. Physiol Behav. 27, 883-886.

Peterson, M.P., Rosvall, K.A., Choi, J.H., Ziegenfus, C., Tang, H., Colbourne, J.K., Ketterson, E.D., 2013. Testosterone affects neural gene expression differently in male and female juncos: A role for hormones in mediating sexual dimorphism and conflict. PLoS One. 8, e61784.

Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29, e45-e45.

Phoenix, C.H., Goy, R.W., Gerall, A.A., Young, W.C., 1959. Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology. 65, 369.

Poulos, M.G., Batra, R., Charizanis, K., Swanson, M.S., 2011. Developments in RNA splicing and disease. Cold Spring Harb Perspect Biol. 3, a000778.

Powers, J.B., 1970. Hormonal control of sexual receptivity during the estrous cycle of the rat. Physiol Behav. 5, 831-835.

Puymirat, J., Etongue-Mayer, P., Dussault, J.H., 1995. Thyroid hormones stabilize acetylcholinesterase mRNA in neuro-2A cells that overexpress the beta 1 thyroid receptor. J Biol Chem. 270, 30651-30656.

Raj, B., Blencowe, B.J., 2015. Alternative splicing in the mammalian nervous system: Recent insights into mechanisms and functional roles. Neuron. 87, 14-27.

Rissman, E.F., 2008. Roles of oestrogen receptors alpha and beta in behavioural neuroendocrinology: Beyond yin/yang. J Neuroendocrinol. 20, 873-879.

Saad, F., Aversa, A., Isidori, A.M., Zafalon, L., Zitzmann, M., Gooren, L., 2011. Onset of effects of testosterone treatment and time span until maximum effects are achieved. Eur J Endocrinol. 165, 675-685.

Sarkissian, M., Winne, A., Lafyatis, R., 1996. The mammalian homolog of suppressor-of-white- apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS. J Biol Chem. 271, 31106-31114.

Simerly, R.B., Chang, C., Muramatsu, M., Swanson, L.W., 1990. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: An in situ hybridization study. J Comp Neurol. 294, 76-95.

Sussman, D., Leung, R.C., Chakravarty, M.M., Lerch, J.P., Taylor, M.J., 2016. Developing human brain: Age-related changes in cortical, subcortical, and cerebellar anatomy. Brain Behav. e00457.

Sutcliffe, J.S., Marshall, K.M., Neill, J.C., 2007. Influence of gender on working and spatial memory in the novel object recognition task in the rat. Behav Brain Res. 177, 117-125.

Swaab, D.F., Chung, W.C., Kruijver, F.P., Hofman, M.A., Ishunina, T.A., 2001. Structural and functional sex differences in the human hypothalamus. Horm Behav. 40, 93-98.

Tohgi, H., Utsugisawa, K., Yamagata, M., Yoshimura, M., 1995. Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus. Brain Res. 700, 245-253.

Tsai, H.W., Grant, P.A., Rissman, E.F., 2009. Sex differences in histone modifications in the neonatal mouse brain. Epigenetics. 4, 47-53.

Tsai, M.J., O'Malley, B.W., 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem. 63, 451-486.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.

Vina, J., Lloret, A., 2010. Why women have more alzheimer's disease than men: Gender and mitochondrial toxicity of amyloid-beta peptide. J Alzheimers Dis. 20 Suppl 2, S527-33.

Vuong, C.K., Black, D.L., Zheng, S., 2016. The neurogenetics of alternative splicing. Nat Rev Neurosci. 17, 265-281.

Washburn, M.P., Koller, A., Oshiro, G., Ulaszek, R.R., Plouffe, D., Deciu, C., Winzeler, E., Yates, J.R.,3rd, 2003. Protein pathway and complex clustering of correlated mRNA and protein expression analyses in saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 100, 3107-3112.

Waters, E.M., Simerly, R.B., 2009. Estrogen induces caspase-dependent cell death during hypothalamic development. J Neurosci. 29, 9714-9718.

Willott, J.F., 2009. Effects of sex, gonadal hormones, and augmented acoustic environments on sensorineural hearing loss and the central auditory system: Insights from research on C57BL/6J mice. Hear Res. 252, 89-99.

Yarbrough, W.G., Quarmby, V.E., Simental, J.A., Joseph, D.R., Sar, M., Lubahn, D.B., Olsen, K.L., French, F.S., Wilson, E.M., 1990. A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat. J Biol Chem. 265, 8893-8900.

Zheng, Y., Kerr, D.S., Darlington, C.L., Smith, P.F., 2003. Unilateral inner ear damage results in lasting changes in hippocampal CA1 field potentials in vitro. Hippocampus. 13, 873-878.

Zhu, Y., Hou, H., Rezai-Zadeh, K., Giunta, B., Ruscin, A., Gemma, C., Jin, J., Dragicevic, N., Bradshaw, P., Rasool, S., Glabe, C.G., Ehrhart, J., Bickford, P., Mori, T., Obregon, D., Town, T., Tan, J., 2011. CD45 deficiency drives amyloid-beta peptide oligomers and neuronal loss in alzheimer's disease mice. J Neurosci. 31, 1355-1365.