Retinoic Acid Synthesis and Degradation: Implications for Proper

RETINOIC ACID SYNTHESIS AND DEGRADATION: IMPLICATIONS FOR PROPER

GERM CELL DEVELOPMENT

TRAVIS KENT

A dissertation submitted in partial fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY School of Molecular Biosciences

DECEMBER 2015

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of TRAVIS KENT find it satisfactory and recommend that it be accepted.

______Michael Griswold, Ph.D., Chair

______Kwan Hee Kim, Ph.D.

______Jon Oatley, Ph.D.

______Terry Hassold, Ph.D.

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank the people who have helped me get to where

I am in my professional career. Much of the work presented in this document would not have been possible without the technical expertise from the Isoherranen and the Hassold-Hunt

Laboratories. I would also like to thank my colleagues, both past and present in the Griswold

Laboratory for their support over the years. My committee has also provided a much needed critical eye to my work. Finally, I would like to thank my friends and family who have provided me with the emotional support needed for me to persevere and thrive at Washington State.

iii

RETINOIC ACID SYNTHESIS AND DEGRADATION: IMPLICATIONS FOR PROPER

GERM CELL DEVELOPMENT

Abstract

by Travis Kent, Ph.D. Washington State University December 2015

Chair: Michael D. Griswold

Spermatogenesis is absolutely essential for mammalian reproduction. This highly organized and tightly controlled process is regulated, in part, by retinoic acid (RA). This essential molecule plays key roles in many aspects of spermatogenesis, namely spermatogonial differentiation, blood-testis barrier (BTB) reorganization, meiotic initiation, and spermiation.

While the importance of RA is well known, the regulation of this molecule has not been well studied. RA availability is directly influenced by its synthesis and degradation, metabolized by the ALDH and CYP26 families respectively. It has recently been published that RA is present in a pulsatile manner across the spermatogenic cycle, but it is not known if these enzyme families play a role in this stage-specific availability of RA. The data presented here provides strong evidence that neither the ALDHs nor the CYP26s are regulated in a cyclic manner, suggesting that the availability of retinaldehyde, the precursor of RA, is responsible of the observed pulsatility. In addition to investigating the stage-specificity of the ALDH and CYP26 enzymes, specific inhibitors were used to investigate the effects on spermatogenesis in a high and low testicular RA environment. The data presented here shows that CYP26 inhibition causes precocious spermatogonial differentiation in the adult mouse and is able to drive synchronous spermatogenesis in the neonatal mouse. Additionally, both low and high testicular RA

environments had abnormal effects on Sertoli cells. After inhibiting RA synthesis, BTB permeability was shown to be increased. Conversely, neonatal animals treated with exogenous

RA were shown to have reduced Sertoli cell number, possibly due to premature cessation of proliferation. Finally, meiotic recombination was shown to be increased and decreased in a low and high testicular RA environment, respectively. These data highlight the importance of proper

RA homeostasis within the testis, and illuminate some of the consequences of abnormal RA levels. Taken all together, the data presented in this work show the importance of understanding the expression and function of the ALDH and CYP26 enzymes in the mammalian testis.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

DEDICATION ...... xii

CHAPTER 1: Checking the Pulse of Vitamin A Metabolism and Signaling during Mammalian

Spermatogenesis

1. TITLE PAGE ...... 2

2. ABSTRACT ...... 3

3. INTRODUCTION ...... 4

4. SPERMATOGENESIS ...... 5

5. VITAMIN A METABOLISM ...... 6

6. THE ROLE OF RA DURING SPERMATOGENESIS ...... 7

a. Spermatogonial differentiation ...... 7

b. Meiosis ...... 10

c. Blood-testis barrier (BTB) ...... 11

d. Spermiogenesis and Spermiation ...... 13

7. WHAT IS THE CAUSE OF STAGE-SPECIFIC RA RESPONSE? ...... 15

a. Retinoid metabolism ...... 15

b. Retinoid signaling ...... 16

c. Retinoid transport and storage ...... 17

8. CONCLUSIONS...... 18

9. AKNOWLEDGEMENTS...... 18

10. AUTHOR CONTRIBUTION ...... 19

11. CONFLICTS OF INTEREST ...... 19

12. REFERENCES AND NOTES ...... 19

13. FIGURES ...... 28

CHAPTER 2: ALDH enzyme expression is independent of the spermatogenic cycle and their inhibition causes misregulation of murine spermatogenesis

1. TITLE PAGE ...... 32

2. ABSTRACT ...... 33

3. INTRODUCTION ...... 34

4. METHODS AND MATERIALS ...... 38

5. RESULTS ...... 48

6. DISCUSSION ...... 54

7. AKNOWLEDGEMENTS...... 60

8. REFERENCES ...... 61

9. FIGURES ...... 69

10. TABLES ...... 82

CHAPTER3: Increased testicular RA has abnormal effects on spermatogenesis

1. TITLE PAGE ...... 122

2. ABSTRACT ...... 123

3. INTRODUCTION ...... 124

vii

4. METHODS AND MATERIALS ...... 127

5. RESULTS ...... 133

6. DISCUSSION ...... 137

7. AKNOWLEDGEMENTS...... 141

8. REFERENCES ...... 142

9. FIGURES ...... 147

10. TABLES ...... 157

CHAPTER 4: Conclusions and Future Directions

1. CONCLUSIONS...... 159

2. FUTURE DIRECTIONS ...... 164

3. REFERENCES ...... 167

viii

LIST OF TABLES Page CHAPTER 2

1. Table 1. Biological functions upregulated after 8 days WIN 18,446 treatment ...... 82

2. Table 2. Biological functions downregulated after 8 days WIN 18,446 treatment ...... 83

3. Supplemental Table 1. Genes upregulated after 8 days of WIN 18,446 treatment ...... 84

4. Supplemental Table 2. Genes downregulated after 8 days of WIN 18,446 treatment...... 86

5. Supplemental Table 3. Transcriptional changes of meiotic genes associated with 8

days of WIN 18,446 treatment ...... 115

6. Supplemental Table 4. Transcriptional changes of tight junction genes associated with

8 days of WIN 18,446 treatment ...... 117

7. Supplemental Table 5. Transcriptional changes of vitamin A metabolism and

signaling genes associated with 8 days of WIN 18,446 treatment ...... 119

8. Supplemental Table 6. Transcriptional changes of steroidogenic genes associated with

8 days of WIN 18,446 treatment ...... 120

CHAPTER 3

1. Table 1. Exogenous RA treatment reduces the recombination rate in testes with

synchronized spermatogenesis ...... 157

LIST OF FIGURES Page CHAPTER 1

1. Figure 1. Overview of Murine Spermatogenesis ...... 28

2. Figure 2. Vitamin A metabolism ...... 29

3. Figure 3. Stage-specificity of RA activity ...... 30

CHAPTER 2

1. Figure 1. ALDH enzymes are differentially localized in the neonatal murine testis ...... 69

2. Figure 2. ALDH enzymes locate to different cell types in the adult murine testis ...... 71

3. Figure 3. ALDH1A1 and ALDH1A2 levels do not vary in a manner similar to RA ...... 73

4. Figure 4. ALDH activity does not undergo cyclic changes ...... 75

5. Figure 5. Animals treated with WIN 18,446 have lowered testicular RA levels ...... 76

6. Figure 6. Blood-testis barrier permeability is adversely affected in animals treated

with WIN 18,446 ...... 77

7. Figure 7. WIN 18,446 treatment increases recombination rate ...... 78

8. Figure 8. WIN 18,446 induces meiotic defects...... 79

9. Supplemental Figure 1. ALDH Western Blots ...... 81

CHAPTER 3

1. Figure 1 Polyclonal antibodies show specificity to CYP26 enzymes...... 147

2. Figure 2: CYP26 localization in the neonatal murine testis ...... 148

3. Figure 3. CYP26 enzymes locate predominantly to PTM cells in the adult murine

testis ...... 150

4. Figure 4. Talarozole is cleared very quickly from the serum ...... 152

5. Figure 5 Talarozole treatment of adult male mice results in misregulation of

spermatogonial differentiation ...... 153

6. Figure 6 Neonatal exposure to talarozole drives synchronous spermatogenesis ...... 155

7. Figure 7 Exogenous neonatal RA treatment reduces Sertoli cell proliferation ...... 156

DEDICATION This dissertation is dedicated to my parents, Ray and Connie Kent, who are the most supportive

people in my life and have convinced me that I can achieve anything I set my mind to.

xii

CHAPTER 1 CHECKING THE PULSE OF VITAMIN A METABOLISM AND SIGNALING

The following chapter is formatted in accordance with guidelines of the Journal of Developmental Biology, and is currently in press.

It should be noted that since the publication of this manuscript, Hogarth et al. 2015 submitted a paper showing that RA levels are, in fact, present in a pulsatile manner across the spermatogenic cycle.

TITLE Checking the Pulse of Vitamin A Metabolism and Signaling during Mammailan Spermatogenesis

AUTHORS Travis Kent and Michael D. Griswold *

AFFILIATIONS School of Molecular Biosciences and the Center for Reproductive Biology, Washington State University, Pullman, WA 99164, USA; E-Mail: [email protected]

*Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-509-335-2240. Received: 15 January 2014; in revised form: 18 March 2014 / Accepted: 18 March 2014 / Published: 21 March 2014

ABSTRACT

Vitamin A has been shown to be essential for a multitude of biological processes vital for mammalian development and homeostasis. Its active metabolite, retinoic acid (RA), is important for establishing and maintaining proper germ cell development. During spermatogenesis, the germ cells orient themselves in very distinct patterns, which have been organized into stages.

There is evidence to show that, in the mouse, RA is needed for many steps during germ cell development. Interestingly, RA has been implicated as playing a role within the same two

Stages: VII and VIII, where meiosis is initiated and spermiation occurs. The goal of this review is to outline this evidence, exploring the relevant players in retinoid metabolism, storage, transport, and signaling. Finally, this review will provide a potential model for how RA activity is organized across the murine stages of the spermatogenic cycle.

Keywords: retinoic acid; Vitamin A; testis; spermatogenesis; pulse

1. Introduction

Retinoic acid (RA) plays a vital role in many different developmental processes in mammals. Embryonically, RA is important in organogenesis, limb-bud development, proper neuronal development, and germ cell fate in the developing gonad. In the urogenital ridge, RA signaling within the embryonic ovary is responsible for fetal entry of oogonia into meiosis while

RA degradation in the embryonic testis opposes this action [1,2], although there has recently been some debate on this front [3,4]. Postnatally, RA is an important molecule for the proper function of many organs, such as skin, lung, kidney, liver, and testis function.

Both high and low levels of RA have been shown to cause aberrant male germ cell development. Investigating the role that RA plays during spermatogenesis is vital to gain a better understanding of this complex biological process, but there are also practical applications for furthering this research. Currently, fifteen percent of couples in the United States suffer from infertility [5]. In approximately half of these diagnoses, the cause can be attributed to the male partner [6]. Unfortunately, the root of male infertility, in most cases, is unknown. Abnormal levels of RA have been associated with sterility [7], therefore, understanding the mechanism of control of RA within the testes will provide a better understanding of the players involved in spermatogenesis and has the potential to provide therapeutic options for those suffering from infertility. In addition, men are seeking to take a more active role in their reproductive health.

Unfortunately, there is an appalling gap in healthcare equality when it comes to contraception.

Women have a variety of contraceptive options, while the only impermanent option available to men is condoms. Because vitamin A metabolism plays such a crucial role in spermatogenesis, perturbing RA synthesis and signaling is an excellent target for non-hormonal, male contraception.

In the following sections, this review will provide a brief overview of both spermatogenesis and vitamin A metabolism, and will summarize the current evidence supporting the hypothesis that RA is the master regulator of the cycle of the seminiferous epithelium.

2. Spermatogenesis

Spermatogenesis is a tightly controlled process that can be divided into three distinct phases: (1) mitosis of spermatogonia, (2) meiotic division of spermatocytes, and (3) the morphological transformation of haploid spermatids to mature spermatozoa (Figure 1A). Diploid spermatogonia undergo several rounds of mitosis to both maintain a healthy stem cell pool and amplify the population of cells committed to undergoing meiosis. The spermatogonial population consists of both undifferentiated and differentiating spermatogonia, and the commitment to differentiate is known to be under the control of RA. Spermatocytes undergo meiosis for the purpose of halving their chromosomal number and ensuring genetic diversity. The first meiotic prophase is elongated during mammalian spermatogenesis when compared to the second, taking approximately twelve days in the mouse. RA is hypothesized to play a role during the initiation of this process. Finally, haploid spermatids undergo gross morphological changes, deemed spermiogenesis, before they are released as mature spermatozoa into the lumen of the seminiferous epithelium. Evidence suggests that both the radical morphological changes, as well as the release of the spermatozoa into the tubule lumen are under control of RA signaling. These spermatozoa are then shuttled to the rete testes where they are released into the epididymis for further maturation.

As germ cells progress through spermatogenesis, they travel from the basement membrane of the seminiferous tubule, where they start as undifferentiated spermatogonia, to the lumen, where they are released as spermatozoa. The columnar Sertoli cells are physically

intertwined with the developing germ cells and provide support, nutrients, and protection (Figure

1B). Much of our current understanding of mammalian spermatogenesis has been derived from studies in rodents and this review will primarily focus on data generated using mouse models. In the mouse, it takes approximately 35 days for an undifferentiated spermatogonium to differentiate, progress through meiosis, undergo spermiogenesis, and be released into the lumen of the tubule [8].

Spermatogenesis takes place in a very organized manner within the seminiferous tubule.

In a normal adult murine testis, specific cell types are always observed together and these associations have been classified into stages [8] (Figure 1C). For example, upon examining a cross section of a seminiferous tubule, one will always observe spermatozoa being released into the lumen when round spermatids, preleptotene spermatocytes, and differentiating spermatogonia are present. Every tubule that contains these properties is a Stage VIII tubule in the mouse testis. If we were to look at a single cross section of a seminiferous tubule over time, we would see this tubule transition through all twelve spermatogenic stages over the course of

8.6 days [8], known as the spermatogenic cycle. In order to continuously produce sperm, spermatogenesis occurs in an asynchronous manner; i.e., all stages of the spermatogenic cycle are represented throughout the testis at any given moment. Current evidence indicates that RA function is essential in two of the twelve stages of murine spermatogenesis: Stages VII and VIII.

3. Vitamin A Metabolism

RA is vital for many different steps during spermatogenesis but cannot be readily transported throughout the body. Retinol (ROL), however, can be ingested through the diet and shuttled easily through the blood stream in complex with retinoid binding proteins (RBPs). When

ROL reaches its target tissue, it is transported into the cell via the transmembrane receptor,

STRA6, in tandem with the retinol binding protein RBP4, and can either be stored as retinyl esters by Lecitin retinol transferase (LRAT) or metabolized [10] (Figure 2). The metabolism of

ROL to RA is a two-step enzymatic reaction, the first of which is the synthesis of retinaldehyde

(RAL) via retinol dehydrogenase (RDH) enzymes. This reaction is thought to be the rate limiting step of vitamin A metabolism and is reversible [10]. The second is the conversion of RAL to RA, catalyzed by retinaldehyde dehydrogenase (RALDH) enzymes.

Once RA is synthesized, it can either be degraded by the cytochrome P450, family 26

(CYP26) enzymes into inert metabolites, or it can bind to the retinoic acid receptors (RARs), which then dimerize with retinoid X receptors (RXRs) [10]. RAR/RXR dimers bind to retinoic acid response elements (RAREs) located throughout the genome and induce differential expression of specific genes [11]. Current data indicate that RA signaling occurs in a stage- specific manner during the spermatogenic cycle.

4. The Role of RA during Spermatogenesis

4.1. Spermatogonial Differentiation

The first step of spermatogenesis is the differentiation of spermatogonia. In the mouse testis, the undifferentiated A spermatogonial population continuously divide in order to repopulate the testis, but a subset of these cells differentiate into A1 spermatogonia, known as the A to A1 transition, every 8.6 days. The A1 spermatogonia express KIT, the most well-known marker of differentiating spermatogonia, along with STRA8. These A1 spermatogonia undergo six mitotic divisions before entering meiosis [12]. In the adult mouse testis, the A to A1 transition takes place during Stage VIII.

Vitamin A is essential for spermatogonial differentiation. When male rodents are made vitamin A deficient (VAD), spermatogenesis is halted [13,14]. The only cell types present in the

seminiferous epithelium of these animals are undifferentiated, KIT-negative spermatogonia and

Sertoli cells [13-15]. When vitamin A, in the form of ROL or RA, is reintroduced, the spermatogonia differentiate simultaneously, becoming both KIT- and STRA8-positive [13,14].

There is sufficient evidence to indicate that RA is directly responsible for driving the expression of Stra8 within the testis [16,17] and a growing body of data to suggest that it also regulates the transcription and translation of Kit [16,18,19]. Furthermore, spermatogenesis reinitiates normally, albeit in a synchronous manner [14,20], i.e., only a few stages of the cycle are present and spermiation occurs throughout the entire testis simultaneously. Transgenic models have also been utilized to investigate the A to A1 transition in the absence of vitamin A in the juvenile testis. Both Lrat- and Rbp4-null mice, while perfectly fertile on a normal diet, become VAD much quicker than their wild type counterparts, likely due to depleted retinoid stores and aberrant transport capabilities [21,22]. The juvenile testes of both these VAD transgenic animals display a halt in spermatogenesis at spermatogonial differentiation [21,22]. Taken together, these data support the idea that vitamin A, or a downstream metabolite, is responsible for the A to A1 transition.

Results similar to what was observed in VAD testes were also seen when RALDH was inhibited either chemically or genetically. When animals were treated with an RALDH inhibitor,

WIN 18,446, their testes were morphologically similar to that of a VAD animal, i.e., the only cell types present within the tubules were undifferentiated spermatogonia and Sertoli cells [23,24].

When animals treated with WIN 18,446 were subsequently dosed with exogenous RA, spermatogenesis resumed normally but, again, in a synchronous manner [23]. A separate study examined testes from mice with Sertoli cell- specific deletions of Aldh1a1, Aldh1a2, and

Aldh1a3 (Raldh1-3), thereby eliminating RALDH activity in these cells [25]. The testes of these

mice also exhibited a VAD-like phenotype, and when these animals were exposed to exogenous

RA, spermatogenesis resumed, not only through the first wave, but also through subsequent waves. [25]. All of these RA-depleted systems provide strong evidence indicating a role for RA during spermatogonial differentiation. The Raldh1-3 Sertoli cell-specific deletion study demonstrates that the A to A1 transition requires RA to be synthesized by Sertoli cells, but only for the first spermatogenic wave.

Ablation of Rdh10 in Sertoli cells of mice also causes a halt in spermatogenesis at the A to A1 transition, although the phenotype is more pronounced when Rdh10 is removed from both

Sertoli and germ cells [26]. Consistent with previous reports, the phenotype is rescued in a synchronous manner when retinoids were administered exogenously [26]. Interestingly, spermatogenesis is restored to normal in the absence of retinoid treatment after approximately 3 weeks in the Rdh10-deficient mice [26], indicating that RDH10 is required for the first and possibly second wave of spermatogenesis but is dispensable for subsequent waves. Because RA plays a role in the maintenance of spermatogenesis, it is probable that expression of a separate

RDH enzyme is responsible for recovered fertility.

Furthermore, aberrant RA signaling inhibits spermatogonial differentiation. When RARγ is deleted either globally or specifically in spermatogonia, a VAD-like phenotype is observed in mice aged 10 weeks or older [27]. Ablation of both RARγ and RARα exacerbates this phenotype

[27]. This indicates that RA signaling within the spermatogonial population is absolutely essential for differentiation to take place.

Finally, exogenous RA has been used to better understand the regulation of the A to A1 transition. Injection of exogenous RA into a neonatal mouse younger than 6 days postpartum

(dpp) is sufficient to stimulate simultaneous spermatogonial differentiation, resulting in a

synchronized testis [28,29]. Interestingly, it becomes impossible to synchronize spermatogenesis in this manner in mice after 8 dpp. What is special about an 8 dpp testis? In the mouse, this corresponds with the first appearance of preleptotene spermatocytes. It is possible that, after the first wave of spermatogenesis, the preleptotenes are the source of RA for the undifferentiated spermatogonia, and retinoid metabolism in the Sertoli cells is no longer required for differentiation. This theory is supported by the observation that spermatogonia in the testes of

Raldh1-3 Sertoli cell-specific knockout mice are capable of differentiating continuously following a single dose of exogenous RA [25].

4.2. Meiosis

Meiosis is the process by which diploid germ cells undergo one round of chromosomal duplication and two rounds of cell division, resulting in four genetically dissimilar haploid daughter cells. Meiosis takes approximately a cycle and a half of the seminiferous epithelium to fully complete in both mice and rats. In adult mice, meiotic initiation takes place during Stage

VII.

Vitamin A plays a key role during male meiosis, with most of the current evidence derived from studies involving the expression and function of Stra8. This gene has been shown to be stimulated in the presence of RA—in fact Stra8 stands for “stimulated by retinoic acid, gene 8”—and has been considered, for many years, to be the classical RA responsive gene

[17,30]. Additionally, inhibition of RADLH activity in mice, via treatment with WIN 18,446 both in vitro and in vivo, was shown to suppress expression of Stra8 [23,31]. Stra8-deficient germ cells fail to properly undergo meiosis in mice [32,33], indicating that Stra8 is vital for normal meiotic progression.

Additional evidence to support a role for RA in the regulation of meiosis has been generated using the embryonic murine gonad as a model. Exogenous RA stimulated the expression of well characterized markers of meiosis, Scp3, Dmc1, and γH2afx, in the embryonic testis [34]. Moreover, inhibition of CYP26 enzymes with ketoconazole in the fetal testis resulted in an up-regulation of Stra8, Scp3, and Dmc1, and the germ cells took on morphological characteristics consistent with meiotic germ cells [1,34]. When fetal testes were treated with both ketoconazole and a pan-RAR antagonist, an induction of meiosis was not observed [1]. In a similar study, larval testes from several species of frogs were treated with exogenous RA or

CYP26 inhibitors, and in both cases, leptotene spermatocytes were observed in the treated testes but not in the vehicle controls [35]. Moreover, when larval frog testes were cultured with either an RALDH inhibitor or an RAR antagonist, formation of leptotene spermatocytes was not observed [35]. Both of these studies implicate RA—specifically RA signaling—as being vital for meiotic progression.

Complementary evidence implicating RA as being responsible for meiotic initiation has also stemmed from studies of mice deficient in CYP26B1. A three-fold increase in RA-induced stimulation of a reporter construct was noted in the embryonic testis of Cyp26b1-null mice, indicative of higher RA levels [2]. An increase in Stra8 and Scp3 expression was also observed as well as the presence of cells morphologically similar to pachytene spermatocytes by embryonic day 16.5 [2,34]. Taken together, these data indicate that both RA accumulation and signaling are sufficient to drive meiosis.

4.3. Blood-Testes Barrier (BTB)

Vitamin A has been implicated in playing a vital role in the maintenance and reorganization of the blood-testis barrier (BTB), which is established by tight-junctions between

adjacent Sertoli cells, forming two compartments for the germ cells to reside within: basal and adluminal. One purpose of the BTB is to provide an immune privileged environment for the developing germ cells. During and after meiosis, the germ cells produce antigenic proteins that are not produced anywhere else in the body and are, therefore, vulnerable to attack and clearance by the immune system. Diploid spermatogonia reside in the basal compartment while spermatocytes and spermatids reside in the adluminal compartment. As differentiating spermatogonia enter into meiosis, they must pass through the BTB in order to reside in an immune privileged environment. During Stage VIII, new tight-junctions are formed on the basal side of the preleptotene spermatocyte as the old tight-junctions on the adluminal side of the cell are degraded [36].

The integrity of the BTB is vital for fertility. When Ocln, a gene known to be essential for the BTB, was deleted in mice, massive germ cell loss was observed in the resulting animals

[37]. Since then, it has been shown that RA induces expression of Ocln and Zo1, BTB genes encoding proteins that play crucial roles in tight-junctions, in vitro and in vivo respectively

[38,39]. Tight-junction formation was stimulated by RA in cultured primary Sertoli cells isolated from a 20 dpp mouse [40]. In VAD mice, misregulation of genes known to be integral to the

BTB, such as Ocln, Cldn11, and Tjp1, were observed via quantitative PCR [41]. In these same animals, OCLN and CLDN11 protein were abnormally localized in the absence of vitamin A

[41]. Together, these data suggest that vitamin A is responsible for the appropriate expression and localization of vital BTB proteins.

Investigation of Rara-null mice has provided further evidence that RA is important for

BTB integrity. Testes from these animals exhibited aberrant cell-cell interactions as well as a delay in incorporation of ZO1 [42]. A separate study investigated BTB function in adult mice

that over-expressed a dominant-negative form of RARα specifically in Sertoli cells [38]. Five days after lentiviral injection, which was used to deliver the transgene, spermatocytes and spermatids underwent apoptosis, but only in Stages VII-XII [38]. Furthermore, ZO1 was downregulated in Stages VII-XII, and a biotin permeability assay, which is used to assess the integrity of the BTB, showed that the barrier was compromised in these same stages [38]. Finally, the authors noted that the Sertoli cells appeared to have detached from the basement membrane of the seminiferous tubule in Stages VII-XII [38]. Interestingly, the investigators in this study examined the testis five days after lentiviral injection and observed all problems taking place in

Stages VII-XII. It takes approximately 4.6 days for a Stage VII tubule to progress to a Stage XII tubule [8], indicating that RA signaling within the Sertoli cell only occurs during Stage VII. In total, these data provide strong evidence that RA signaling via the Sertoli cells at Stage VII of the cycle of the seminiferous epithelium is important for maintaining BTB integrity.

4.4. Spermiogenesis and Spermiation

Spermiogenesis occurs following meiosis when the newly formed round spermatids undergo gross morphological changes to become elongated spermatozoa. These spermatozoa are then released into the lumen of the seminiferous tubule during Stage VIII of the cycle, in a process called spermiation. Perturbations in RA metabolism and signaling have shown defects in both of these processes, but the extent to which RA plays a role has not been sufficiently examined.

Spermiation occurs abnormally in VAD rodents. In both Rbp4- and Lrat-null mice fed a

VAD diet, spermatids were retained well past Stage VIII [21,43], and in VAD rats, spermatozoa were retained in many tubules as late as Stage XI [44]. In addition, when RA synthesis was ablated in Sertoli cells of mice, spermatids failed to properly align and were retained within the

seminiferous epithelium later than normal [25]. These data show that the presence of RA, particularly in Sertoli cells, is essential for proper release of spermatozoa into the lumen of the tubule.

There is also evidence to indicate that RAR signaling is required for proper spermiogenesis and spermiation. When a dominant-negative form of RARα was over-expressed in Sertoli cells of adult mice, spermatids failed to release during Stage VIII and remained present at Stage IX [38], similar to what was observed in the RA deficient rats and mice. When Rara was globally excised in mice, the resulting testes showed a developmental arrest at step 8–9 spermatids during the first wave of spermatogenesis as well as a failure of these spermatids to properly align for release one cycle later during Stage VIII [45], but fertility was restored in these animals when Rara was overexpressed in haploid spermatids [46]. A separate study from the same laboratory demonstrated that RARα-deficient stem cells were capable of repopulating a germ cell-depleted wild type testis but produced spermatozoa exhibiting abnormal morphology, such as blunted heads [46]. Additionally, the researchers noted atypical chromatin condensation, reduced total cell number, and irregular cellular associations in the spermatids of these animals

[46]. When RAR activity is chemically inhibited via a pan-RAR antagonist (BMS-189453), defects in spermiogenesis and spermiation also occur. Spermatids failed to properly align and release during Stages VIII and IX [47], and one month after a week of treatment with BMS-

189453, some tubules were missing entire layers of germ cells [47]. All of these data strongly suggest that retinoid signaling via RARα is playing a crucial role in both spermatid maturation and release into the lumen of the seminiferous tubule.

Finally, defects in spermiation were observed in the Rxrb-deficient mouse. When this transcription factor was ablated specifically in Sertoli cells, spermatids failed to align on the

luminal side of the seminiferous tubule and were not released through Stages IX and X [43].

Significantly more TUNEL-positive, apoptotic cells were also observed in the testes of these animals, which the authors hypothesized as being the result of phagocytized spermatids that were unable to be released into the tubule lumen [43]. These data indicate that signaling through

RXRβ in Sertoli cells is vital to proper spermiation. As both RARα and RXRβ have been localized to Sertoli cells [48], it is likely that these two transcription factors heterodimerize to facilitate spermiation during Stage VIII.

5. What is the Cause of Stage-Specific RA Response?

As detailed above, RA is vital for many different steps in spermatogenesis and, interestingly, all of these processes occur during the same spermatogenic stages (Figure 3).

Spermatogonial differentiation, meiotic initiation, reorganization of the BTB, and spermiation all occur during Stages VII and VIII of the spermatogenic cycle.

This observation strongly indicates that RA activity is only important during a very small window of spermatogenesis. It is, at this point, unclear if RA itself is regulated in a pulsatile manner across the spermatogenic cycle, or if RA is present throughout the cycle, but another level of control renders its presence inert until the cells within the tubule are ready to respond. It is likely that these two ideas are not mutually exclusive. In order to further tease this question apart, stage-specificity of relevant players associated with RA metabolism, signaling, transport, and storage must be assessed in order to determine how all of these processes could contribute to the hypothesized pulse of RA.

5.1. Retinoid Metabolism

If RA is only present during Stages VII and VIII, how is this phenomenon regulated? Is

RA synthesis or degradation responsible for generating this pulse? Localization studies from

mice (reviewed in [31]) have provided some clues. Aldh1a1 has been observed in all Leydig and

Sertoli cells [48]. Aldh1a2 transcript is expressed in late spermatocytes and early spermatids but does not appear to be stage-specific [48] and a separate protein localization study has confirmed localization of ALDH1A2 to early spermatids but not late spermatocytes [49]. Finally, Aldh1a3 mRNA was only detected at low levels in Leydig cells [48]. Another retinoid metabolizing gene that may be of biological significance is Rdh11, which was shown to have variable expression across the spermatogenic stages and is present in pachytene spermatocytes [50]. The retinoid degrading genes, Cyp26a1, Cyp26b1, and Cyp26c1, all localize to peritubular myoid cells, which form the outer layer of the seminiferous tubule, in all stages [48]. CYP26B1 enzyme has also been noted in the peritubular myoid cells [49].

Taken together, this localization data can provide a working hypothesis. If RA metabolism within the seminiferous tubule is, in fact, what is responsible for maintaining spermatogenesis, then logically, the enzymes responsible for either the synthesis or the degradation, or both, would be present in the testis in a stage-specific manner. This, however, is not the case [48,49]. It is possible that RAL availability is indirectly responsible for generating this hypothesized pulse of RA, as preliminary data suggests that Rdh11 is present in a stage- specific manner [50]. Further investigation needs to be performed to determine if protein localization and enzymatic activity of the vitamin A metabolizing enzymes fluctuates with the cycle of the seminiferous epithelium in order to generate a cyclic RA pulse.

5.2. Retinoid Signaling

If, it turns out, RA metabolism is not—or only partially—responsible for the observed

RA response, then another method of regulation must exist. One candidate may be the cell’s ability to respond to RA. Again, localization studies have provided insight into whether there is

stage-specificity associated with RA signaling molecules (reviewed in [31]). Rara and Rxrb transcript and protein were detected in Sertoli cells of all tubules, while Rarb and Rxra transcripts were localized to round spermatids, specifically in Stages VII and VIII [48]. A separate study in adult rats found that RARα is expressed in spermatids [51]. Rarg mRNA was detected at the periphery of all tubules, while Rxrg mRNA was localized to some round spermatids in a fraction of Stage VII and VIII tubules [48]. RARβ is expressed in Sertoli cells, while RXRα and RXRγ are expressed in the majority of germ cells [52]. If retinoid signaling is responsible for the stage-specific RA response, it is probably the result of signaling via RARβ,

RXRα, or RXRγ, or more likely, a combination of the three. Separate studies reported that Rarb-,

Rxrg-, and Rarb/Rxrg-null animals have no phenotype associated with sterility [48,53,54]. It is possible that ablation of these genes caused a change in expression of the other RARs and RXRs to compensate for the loss. Moreover, these genes could be acting in a non-canonical manner.

Regardless, more conclusive localization studies need to be conducted to flesh out currently reported inconsistencies.

5.3. Retinoid Transport and Storage

Retinoid transport and storage is another potential cause for variable RA levels across the spermatogenic cycle. Stra6 is expressed specifically in Stages VI and VII [55], but no phenotype associated with fertility, or even aberrant spermatogenesis, was reported for Stra6-null animals

[56]. The localization of Crabp genes, which encode for proteins responsible for cellular binding of retinoids, within the testis was also investigated. Crabp1 localized to both undifferentiated and differentiating spermatogonia in the rat and the mouse [48,57], while CRABP2 was localized to the testis in rats via immunoblotting, though more specific localization data were not pursued

[58]. Lrat is expressed in round spermatids during Stages II-VI and Rbp1 transcript was detected

in Sertoli cells during stages X-XI [48]. The localization patterns of both Lrat and Rbp1 support the hypothesis that stage-dependent transport and storage of retinoids plays a role in cyclic RA availability within the testis.

A definitive investigation of whether retinoid metabolism, signaling, transport, or storage—or a combination of these—is responsible for the observed stage-dependent response to

RA during spermatogenesis is still required. Understanding how this response is generated will be pivotal in moving forward the fields of both spermatogenesis and retinoid biology.

6. Conclusions

RA plays key roles in neonatal and adult spermatogenesis. In the neonatal murine testis, it has been hypothesized as being responsible for establishing the spermatogenesis in an asynchronous manner [59]. It also plays a role in many aspects of adult spermatogenesis, being vital for spermatogonial differentiation, meiotic initiation, and reorganization of the BTB, as well as release of spermatozoa into the lumen of the seminiferous epithelium. Importantly, all of these processes occur during the same two stages of spermatogenesis: Stages VII and VIII. This strongly suggests that RA is only necessary during these two stages, and other stages progress in either the absence of RA or these cells temporarily lack the ability to respond to RA.

Because RA is known to play such a vital role in so many different aspects of mammalian biology, it is crucial that we understand the action and regulation of this integral biological molecule, as it can have important implications in many different disciplines. Hopefully this review will elucidate avenues of future research in not only the field of spermatogenesis, but also in retinoid biology and development.

Acknowledgments

The authors would like to acknowledge Dr. Cathryn Hogarth for her critical review of this manuscript. This work was supported by the NIH Grant HD 10808 and U54 HD 042454.

Author Contribution

T Kent and M Griswold conceived these ideas. T Kent wrote the manuscript. T Kent and M

Griswold edited the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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(http://creativecommons.org/licenses/by/3.0/).

FIGURES

Figure 1. Overview of Murine Spermatogenesis. (A) Schematic representing the types of germ cells present within the seminiferous tubule. (B) Schematic representing the organization of the adult seminiferous tubule. Cell types in B correspond with those in A. Gray blocks represent

Sertoli cell nuclei, while the white spaces between cells represent Sertoli cell cytoplasm. (C)

Diagram depicting the twelve cellular associations present in murine spermatogenesis. The red arrow represents spermatogonial differentiation. (Figure 1C was adapted from [9]).

Figure 2. Vitamin A metabolism. This diagram represents vitamin A metabolism as it relates to spermatogenesis. Retinol (ROL), which is transported in complex with RBP4, is either stored or metabolized to RA via a two-step enzymatic process. Retinoic acid (RA) is then either degraded or utilized to allow transcription factors, in complex with cellular retinoic acid binding protein

(CRABP), to stimulate gene expression. Relevant enzymes are labeled in red. Transcription factors are labeled in orange. Retinoid binding proteins are labeled in green. Membrane bound transporters are labeled in blue.

Figure 3. Stage-specificity of RA activity. This figure briefly outlines the relevant data that provide evidence supporting Stage-specificity of RA activity during Stages VII and VIII. RA has been shown to be vital during spermatogonial differentiation, meiotic initiation, blood-testis barrier (BTB) reorganization, spermiogenesis, and spermiation. All of these steps of spermatogenesis take place during Stages VII and VIII indicating that RA activity is only required during these two stages. (Figure adapted from [9]).

CHAPTER 2

ALDH ENZYME EXPRESSION IS INDEPENDENT OF THE SPERMATOGENIC CYCLE

AND THEIR INHIBITION CAUSES MISREGULATION OF MURINE

SPERMATOGENESIS.

The following chapter is formatted in accordance with guidelines of the Biology of

Reproduction, and is currently in review.

ALDH enzyme expression is independent of the spermatogenic cycle and their inhibition causes

misregulation of murine spermatogenesis.1

Travis Kent3, Samuel L. Arnold4, Rachael Fasnacht3, Ross Rowsey3, Debra Mitchell3, Cathryn

A. Hogarth3, Nina Isoherranen4, and Michael D. Griswold2,3

3School of Molecular Biosciences and The Center for Reproductive Biology, Washington State

University, Pullman, Washington, USA.

4Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.

Short title: ALDHs are vital to spermatogenesis, not RA pulses.

Summary sentence: ALDH enzymes are likely not responsible for the regulation of RA cyclicity,

but are vital for both BTB maintenance and meiosis.

Key words: ALDH, testis, meiosis, blood-testis barrier, retinoic acid.

______

1 This research was supported by NIH grants R01 10808 to MDG, U54 HD 42454 to MDG and

NI, R01 GM111772 to NI and CAH. Partial support for this project was funded by T32

GM083864 to TK from the NIGMS and TL1 TR000422 to SA from NCATS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the

National Institute of General Medical Sciences or the National Institutes of Health

2 Corresponding author: Professor Michael Griswold, School of Molecular Biosciences, PO Box

647520, Pullman, WA, 99164 Email: [email protected]

Abstract

Perturbations in the vitamin A metabolism pathway could be a significant cause of male infertility, as well as a target towards the development of a male contraceptive, necessitating the need for a better understanding of how testicular retinoic acid (RA) concentrations are regulated.

Quantitative analyses have recently demonstrated that RA is present in a pulsatile manner along testis tubules. However, it is unclear if the aldehyde dehydrogenase (ALDH) enzymes, which are responsible for RA synthesis, contribute to the regulation of these RA concentration gradients. Previous studies have alluded to fluctuations in ALDH enzymes across the spermatogenic cycle, but these inferences have been based primarily on qualitative transcript localization experiments. Here we show via various quantitative methods that the three well- known ALDH enzymes (ALDH1A1, ALDH1A2, and ALDH1A3), and an ALDH enzyme previously unreported in the murine testis (ALDH8A1), are not expressed in a stage-specific manner in the adult testis, but do fluctuate throughout juvenile development in perfect agreement with the first appearance of each advancing germ cell type. We also show, via treatments with a known ALDH inhibitor, lowered testicular RA levels result in an increase in blood-testis barrier permeability, meiotic recombination, and meiotic defects. Taken together, these data further our understanding of the complex regulatory actions of RA on various spermatogenic events and, in contrast with previous studies, also suggest that the ALDH enzymes are not responsible for regulating the recently measured RA pulse.

Introduction

Vitamin A metabolism is vital for proper spermatogenesis. Precise regulation of the availability of retinoic acid (RA), the active metabolite of vitamin A, is important for spermatogonial differentiation, blood-testis barrier (BTB) function, meiotic initiation, and proper spermiation (reviewed in [1]). However, the extent of the role RA plays in regulating these spermatogenic processes or the enzymes and cell types involved in controlling RA levels within the mammalian testis have yet to be fully elucidated. An enhanced understanding of how RA concentrations are regulated within the testis and the complex effects of this molecule on various events during spermatogenesis could have important clinical implications for the treatment of idiopathic male infertility, as well as the development of a safe, effective, and reversible oral male contraceptive.

Retinol, the alcohol form of vitamin A, is transported via serum throughout the body.

Once it reaches target tissues, retinol is converted to RA by way of a two-step enzymatic process, the last of which is catalyzed by the aldehyde dehydrogenase (ALDH) enzymes [2]. There are three known RA synthesizing ALDHs whose transcripts have been localized within the murine testis: Aldh1a1, Aldh1a2, and Aldh1a3 [3-5]. Thus far, however, reports regarding the localization of these enzymes have been contradictory, incomplete, and focused predominantly on the adult mouse testis. A recent publication reported cell-specific ALDH protein localization in the adult human testis [6], yet the near complete lack of available prepubertal human tissue has meant that the expression and activity of these enzymes during human testis development has remains unclear. A thorough investigation of the ALDH enzymes in both the neonatal and adult testis will help clarify results from contradictory studies and advance our understanding of RA

synthesis in the testis throughout development, using the mouse as a model of mammalian spermatogenesis.

There are now multiple lines of evidence to support the hypothesis that RA gradients exist along testis tubules [4, 5, 7], yet there is no data regarding how these gradients are established. Several transcript localization studies have alluded to ALDH1A2 perhaps regulating testicular RA in a pulsatile manner [4, 5], but no quantitative data exists to support this conclusion. Interestingly, the ALDH isozymes have recently been predicted to contribute differently to total testicular RA levels [6, 8]. While 10-fold more ALDH1A1 protein is present in the murine testis compared to ALDH1A2, ALDH1A2 is expected to contribute 61% of the total RA synthesis in the murine testis [8], while in the human testis, the expected contribution of

ALDH1A2 is lower: just 15% [6]. Notably, these studies were performed on whole testis, not in a stage-specific manner, making it impossible to determine if these differences in isozyme activity contribute to generating RA gradients along testis tubules. A quantitative analysis to measure undulations in ALDH expression and activity along testis tubules is required to determine whether these enzymes are responsible for the proposed RA pulse.

The pulsatility of RA also highlights its importance during spermatogenesis. RA is thought to be vital for several spermatogenic processes, all of which take place when RA levels are highest [1, 7]. The best characterized of these is spermatogonial differentiation, but RA has also been implicated in BTB reorganization, meiotic initiation, and spermiation ([1, 9] and references therein). The BTB is misregulated in mice with aberrant RA signaling in Sertoli cells

[10] and the transcription of Stra8, a gene known to be important for proper meiosis [11, 12], is under the direct control of RA [13, 14]. Genes associated with regulating both BTB formation

and maintenance, and meiotic initiation and execution have also been shown to be misregulated in RA-deficient models [15].

Due to the complex organization of the testis, the direct study of the mechanisms by which RA controls BTB reorganization, meiotic initiation, and spermiation, is difficult. RA- deficient models, as well as their respective RA-rescue models, have been used in an attempt to elucidate the roles of this molecule during spermatogenesis [15-25] but have numerous disadvantages. Animals on a vitamin A-deficient (VAD) diet can still release retinol from stored retinyl esters, so an extended length of time is required for the animals to become RA deficient

[2]. While it is possible to examine spermatogenesis in animals during induced RA deficiency

[16, 20, 21], RA levels were not quantified in these models. As a result, the extent to which dietary induced-deficiency lowers RA levels over time within the testis is unknown, making it difficult to draw conclusions regarding how RA deficiency leads to aberrant spermatogenesis. In addition, once a testis is completely RA deficient, the only germ cells present within the seminiferous tubules are undifferentiated spermatogonia; when RA is reintroduced to this environment, spermatogenesis continues normally, albeit in a synchronous manner [19, 21, 22,

24, 25]. While providing an excellent tool for the study of spermatogonial differentiation, these

RA-deprived models are not useful in determining the effects of RA on other spermatogenic processes, such as BTB reorganization and meiotic initiation, as the relevant cell types are not present in the RA deficient environment.

The goals of this study were to determine 1) the cell types that synthesize RA in the developing post-natal murine testis, 2) if ALDHs are responsible for the generation of RA pulses in the testis, 3) the gene expression changes associated with a lowered RA environment and 4) if any spermatogenic defects are associated with lowered testicular RA. Based on ALDH

localization, protein quantification, and enzyme activity, the results of this study strongly suggest that ALDH availability is not primarily responsible for the regulation of the RA pulse.

Additionally, it is evident that an RA-deficient environment is detrimental to both BTB permeability and meiosis.

Materials and Methods

Animal care and handling

All experiments were conducted using C57BL/6-129 mice following the approval of the

Washington State University Animal Care and Use Committee. Mouse colonies were housed in a temperature- and humidity-controlled environment and food and water was provided ad libitum. Mice were treated as described below and sacrificed by CO2 asphyxiation followed by either decapitation (0-10 days post-partum [dpp]) or cervical dislocation (> 10 dpp). Tissue was dissected from the mouse for further analysis.

WIN 18,446 and RA treatments

Spermatogenesis was synchronized as previously described [22]. Briefly, 2 dpp male mice were treated with 100 μg/g body weight WIN 18,446 (a kind gift from Dr. John Amory from the University of Washington) or vehicle (1% gum tragacanth) for 7 days. On the following day (8th day of treatment, 9 dpp), the animals were either euthanized as WIN 18,446 or vehicle (1% gum tragacanth) only-treated animals or treated with 200 μg all-trans RA (atRA)

(Sigma Aldrich), or vehicle (dimethyl sulfoxide [DMSO]). The animals given injections were then sacrificed at various time points between 1 - 16 days following treatment (induced spermatogenic synchrony) for neonatal analysis or 42 - 49 days for analysis of synchronized spermatogenesis in the adult testis. For the neonatal time points, pooled testis samples (n = 3 per time point) weighing at least 30 μg each (approximately 3 animals for 0-4 days post treatment, 2 animals for 6 days post treatment, and 1 animal for all older time points) were used for ALDH quantification and activity. For each adult animal, one testis was used to determine synchrony and the other was used for ALDH quantification and activity measurements.

To investigate the effects of ALDH inhibition on adult spermatogenesis, adult mice (3-5 months of age) were treated orally with either 125 mg/kg/day WIN 18,446 or vehicle (1% gum tragacanth) for either 1, 8, or 12 days. Animals were euthanized between 0 and 24 hours following their last dose. The testes were then dissected from these animals and used either for

RA quantification, biotin permeability assays, meiotic spreads, or RNA sequencing.

Western Blotting

Western blots were performed using rabbit polyclonal antibodies specific to ALDH1A1

(Abcam plc, ab24343, 0.1 μg/mL), ALDH1A2 (Proteintech Group, 13951-1-AP, 1.3 μg/mL),

ALDH1A3 (Abgent, AP7847a, 2.5 μg/mL), and ALDH8A1 (Santa Cruz Biotechnology, sc-

130686, 0.1 μg/mL). Briefly, equal amounts of adult mouse testis protein was loaded onto and separated via SDS-PAGE (Bio-Rad Laboratories, #456-1084) and transferred to a nitrocellulose membrane. The membrane was then washed briefly with Tris-buffered saline (TBS) (50 mM

Tris Base, 0.9% NaCl, pH 7.5) and blocked with 5% skim milk in TBS-T (TBS + 0.1% Tween-

20) for an hour at room temperature. Antibodies were diluted in 5% skim milk/TBS-T and applied to the membranes for approximately 20 h at 4°C. The membranes were then washed three times with TBS-T for 5 min each at room temperature. Secondary antibody (Cell Signaling

Technology, #7074, 1:1000 dilution) was applied to membranes for one hour at room temperature. Membranes were again washed three times with TBS-T for 5 min each before they were imaged via chemilumenescence. The membranes were exposed between 15 s and 5 min and imaged on Fujifilm LAS-4000.

Immunohistochemistry

Immunohistochemistry was performed as previously described [26] using mouse testis tissue (n = 3) fixed in Bouin’s fixative, embedded in paraffin, and sectioned onto charged glass

slides using the same antibodies used for Western Blotting. Antigen retrieval was achieved using citrate buffer (10 mM, pH 6) at a rolling boil for 5 min. Sections were incubated in primary antibody at a concentration of 0.5 µg/mL (ALDH1A1), 4 µg/mL (ALDH1A2), 2.5

µg/mL (ALDH1A3), or 0.1 µg/mL (ALDH8A1) in 5% normal goat serum/ 0.1% bovine serum albumin in phosphate buffered saline (PBS) (137mM NaCl/2.7mM KCl/10.1mM

Na2HPO4/1.8mM KH2PO4) at 4°C overnight (~16 h). Control sections were incubated without primary antibody. Biotinylated goat-anti-rabbit secondary antibody (Intvitrogen, 956143b) was applied for 1 h at room temperature, following the manufacturer’s instructions. Strepdavidin conjugated horseradish peroxidase (HRP) (Invitrogen, 956143b) was also applied for 1 h at room temperature. Binding was determined by a brown precipitate formed by HRP activity in the presence of 3,3′-diaminobenzidine tetrahydrochloride (Invitrogen, 002020). Sections were counterstained with a 1:3 dilution Harris Heamotoxilin (Sigma-Aldrich, HHS32-1L), dehydrated, and mounted under glass coverslips using DPX mounting media (VWR International, 360294H).

Cell types were determined using nuclear morphology and location within the testis [27].

Generation of subcellular fractions from mouse tissue

Mouse testis S10 fractions containing microsomes and cytosol were separately generated from

48 samples containing pooled testes from neonatal mice (24-50 mg) and 40 samples containing individual testes from adult mice (43-89 mg), using a previously described method [8]. The total protein concentration in each S10 fraction was measured using a BCA assay (Thermo Scientific,

PI-23227).

Mass spectrometric quantification of ALDH enzymes

The expression of ALDH1A1, ALDH1A2, and ALDH1A3 were quantified in testis S10 fractions using LC-MS/MS as described previously with minor modification [8]. Briefly, the

signature peptides used for quantitation were ANNTFYGLAAGLFTK for ALDH1A1,

EEIFGPVQEILR for ALDH1A2, and EEIGGPVQPILK for ALDH1A3. To confirm the identification of each ALDH1A in the assay, a second peptide was monitored for each ALDH protein. These second peptides monitored were VAFTGSTQVGK for ALDH1A1, the peptide

IFVEESIYEEFVK for ALDH1A2, and ELGEYALAEYTEVK for ALDH1A3. A [13C615N2]- lysine labeled ANNTFYGLAAGLFTK peptide was synthesized as an internal standard for

ALDH1A1. The internal standard peptides for ALDH1A2 and ALDH1A3 were synthesized as extended versions of the quantification peptide with the following sequences

VTDDMRIAKEEIFGPVQEILR and EVTDNMRIAKEEIFGPVQPILK respectively. These peptides contained a C-terminal [13C615N2]-arginine and required two cleavages by trypsin to generate the target peptide. Samples were quantified by mass spectrometry using an AB Sciex

5500 qTrap Q-LIT mass spectrometer equipped with an Agilent 1290 UHPLC as described previously [8]. Each tissue sample was digested in triplicate as described [8] and the resulting peak area for each quantitation peptide was normalized to its corresponding internal standard.

The average value of the three digestions was used along with the standard curve for each protein to determine the pmol of enzyme in each sample. The amount of enzyme in each sample was normalized to the total S10 protein (0.08 mg) in each digestion. All data analysis was performed using Analyst (version 1.5.1) (AB Sciex). A signal:noise ratio of 9 was set as the minimum threshold for quantitation.

Mass spectrometric quantification of atRA

The concentrations of atRA in incubations and tissue samples were measured using an AB Sciex

5500 qTrap Q-LIT mass spectrometer equipped with an Agilent 1290 UHPLC as previously described [7, 8]. For quantification, atRA peak areas were normalized to the atRA-d5 internal

standard peak area. All data analysis was performed using Analyst (version 1.5.1) (AB Sciex).

A signal:noise ratio of 9 was set as the minimum threshold for quantitation.

ALDH Activity

To determine atRA formation in testicular S10 protein from mice, the formation of atRA was measured in the testis S10 fractions using previously described methods with a few modifications [8]. Briefly, 5 μg testicular S10 protein individually from each of the mice was incubated with at-retinal at a nominal concentration of 1000 nM in 100 μl of buffer consisting of

750 mM KCl, 50 mM Hepes, and 2 mM NAD+ at pH 8.0. The incubations were initiated with substrate, performed in triplicate, and terminated after 10 min. The incubations were terminated by transferring 75 μL of the incubation into an equal volume of chilled acetonitrile with 100 nM atRA-d5 (internal standard) and analyzed by LC-MS/MS as described above. The measured concentration of atRA in each incubation was used to calculate the pmol of atRA formed per S10 protein (5 μg) in unit time (10 min) to determine the velocity of atRA formation.

Adult mouse testis staging and analysis

The stage distribution of both unsynchronized and synchronized animals was determined using previously established guidelines [27-29]. Stage distribution was determined by analyzing at least 200 tubules from at least two histological cross sections separated by 50 μm in 30 synchronized animals and 10 unsynchronized controls. The midpoint of synchrony, window width, and synchrony factor were determined as previously described [7, 28, 29]. Five animals were excluded from the analysis because samples were lost during preparation or their synchrony factor was less than 3, leaving 25 animals that were included in the analysis.

To determine whether there were statistically significant changes in ALDH levels or activity across the cycle, the samples were divided into three separate bins: before the RA pulse

(Stages II-VII), during the RA pulse (VIII – IX) and after the RA pulse (X-I) [7]. Unpaired two- tailed student t-tests were then conducted comparing the samples in a particular bin to all other samples. This was performed for each of the three bins.

Biotin Permeability Assay

Fresh testes from adult animals treated for 8 days with either WIN 18,446 (n=4) or vehicle (n=4) were used to determine the integrity of the BTB, as previously described [30].

Briefly, one testis from each animal was injected with approximately 10% testis weight with

PBS containing 1 mM of CaCl2, while the other testis was injected with 10 mg/mL of EZ-Link

Sulfo-NHS-LC-Biotin (Pierce, #21335) dissolved in the same solution. The testes were incubated for 30 min in their respective solutions. After incubation, the testes were washed with

PBS twice for 5 min each before they were fixed with Bouin’s fixative for 6 h. The testes were then dehydrated via ethanol wash gradient, mounted in paraffin, and sectioned onto charged glass coverslips. The slides were rehydrated via a graded ethanol wash series and blocked with

5% normal goat serum/ 0.1% bovine serum albumin in 1X PBS. Strepdavidin conjugated horseradish peroxidase (Invitrogen, 956143b) was then applied for 1 h at room temperature.

Binding was determined by a brown precipitate formed by horseradish peroxidase activity in the presence of 3,3′-diaminobenzidine tetrahydrochloride (Invitrogen, 002020). Sections were counterstained with a 1:3 dilution Harris Heamotoxilin (Sigma-Aldrich, HHS32-1L), dehydrated, and mounted under glass coverslips using DPX mounting media (VWR International, 360294H).

A minimum of 200 tubules was counted for each animal and permeability was determined based on infiltration of brown staining into the luminal side of the BTB. An unpaired one-tailed student t-test was used to determine statistical significance between WIN 18,446 and control treated animals.

Meiotic Preparations and Immunostaining

Testes dissected from adult mice treated with either WIN 18,446 (n=4) or vehicle (n=4) for 12 days were placed in PBS. Meiotic preparations were made as described in [31], with one minor modification: instead of dipping the slide in 1% paraformaldehyde, a thin layer was spread across an uncharged glass slide using a glass Pasteur pipette. Slides were allowed to incubate in a humid chamber overnight, dried, washed in 0.4% Photo-flo 200 solution (Kodak professional), air dried, and immunostained.

For immunostaining, slides were blocked using antibody dilution buffer (ADB) (10 mL normal donkey serum (Jackson Immunoresearch, 017-000-001), 3 g OmniPur bovine serum albumin, Fraction V (EMD Millipore, 9048-40-8), 50 μL Triton X-100 and 990 mL PBS, sterile filtered) for an hour at room temperature. Slides were incubated with SYCP3 antibody (Santa

Cruz Biotechnology, sc-74569, at 0.5 μg/mL) and either MLH1 (Calbiochem, PC56, at 1.3

μg/mL) or SYCP1 (Novus Biologicals, nb 300-229, at 10 μg/mL) antibody; MLH1 or SYCP1 primary antibody in ADB was applied to the slide, covered with a glass coverslip, sealed with rubber cement, and incubated at 37°C for approximately 16 h. Following brief ADB wash,

SYCP3 primary antibody (diluted in ADB) was applied for 2 h at 37°C under a parafilm coverslip. At the end of the incubation period, slides were washed twice in ADB, one hour per wash, at room temperature. Alexa Fluor 488-conjugated AffiniPure Donkey Anti-Rabbit secondary antibody (Jackson Immunoresearch Laboratories, Inc., 711-545-152), was then applied to slides before covering with a glass coverslip, sealing with rubber cement, and incubating for approximately 16 h at 37°C. At the end of the incubation, slides were briefly washed with ADB before Cy3-conjugated AffiniPure Donkey Anti-Mouse secondary antibody

(Jackson Immunoresearch Laboratories, Inc., 715-165-150, at 625 μg/mL) was applied with

parafilm coverslip for 45 min at 37° C. Finally, slides were washed in PBS, and mounted with

20 μL of Prolong Gold anti-fade reagent with DAPI (Life Technologies, P36931) using glass coverslips. Slides were dried in the dark and stored at 4°C.

Recombination Analysis

MLH1 foci counts were conducted on 25 pachytene stage cells per animal by two independent scorers who were blinded with respect to animal status (control vs. treated). Minor scoring discrepancies between scorers were resolved, and cells with major discrepancies were discarded. Cells with poor staining or synaptic defects were excluded from analysis. An unpaired two-tailed student t-test was conducted to compare the recombination rate in WIN

18,446 and vehicle treated animals. The number of MLH1 foci per synaptonemal complex (SC) per cell was also assessed. A chi-squared analysis was used to determine if the distribution of

MLH1 foci along the SCs in the WIN 18,446 treated animals differed significantly from those in the vehicle treated animals.

Meiotic Defect Analysis

In a separate analysis, meiotic defects were assessed by assaying 50 pachytene stage cells per animal, using anti SYCP1 and SYCP3 antibodies to detect SCs. Cells were binned into four categories, as described previously [32]: 1) cells with no detectable meiotic defects, 2) cells with minor synaptic defects (e.g., forks, bubbles and gaps), 3) cells with major synaptic defects (e.g., partial or complete asynapsis of homologous chromosomes), or 4) cells with telomeric associations between non-homologous chromosomes. Gaps were defined as a break in SC staining longer than the width of the SC. Forks and bubbles were defined as partial asynapsis, not longer than the width of the SC, at either the distal ends (forks) or internally (bubbles) on the

SC. Major defects were defined as either complete asynapsis of homologous chromosomes or

asynapsis at least one third the length of the total SC (partial asynapsis). The frequency of these events was determined in vehicle treated animals and a chi-squared analysis was performed to determine if the frequency of these events in WIN 18,446 treated animal differed in a statistically significant manner.

RNA Sequencing and analysis

RNA was isolated from the testes of adult mice treated for 8 days with either WIN 18,446

(n=2) or vehicle (n=2) 24 h following the final dose using TRIzol reagent (Invitrogen, 15596-

018), as per provided instructions. Cytoplasmic and mitochondrial rRNA was removed using the

RiboMinus Eukaryote System (Life Technologies, A15020) and the remaining RNA was concentrated using the RiboMinus Concentration Module (Life Technologies, K1550-05), adhering to the provided instructions in both cases. 100 ng of mRNA was fragmented with

RNAse III and used to construct barcoded sequencing libraries with the Ion RNA-seq kit version

2 (Life Technologies, 4475936). Throughout library preparation, AMPureXP beads (Beckman

Coulter Genomics, A63880) were used in place of the standard purification module. Fragmented

RNA and cDNA were purified using 1X concentration of AMPureXP beads. Size selection was also performed using AMPureXP beads, discarding oversized fragments that bound to 0.7X and a final library capture with 0.9X. Resulting libraries had a peak size distribution of 225 bp. The four libraries were sequenced on two Ion P1 semiconductor sequencing chips, resulting in 126 million reads with a mean readlength of 115bp. 80% of the reads mapped to the Mus musculus reference version 38, using CLC Genomics Workbench 7.5.1. To determine whether genes were either up- or downregulated after WIN 18,446 treatment, several parameters were met: the difference between the average RPKM values of the WIN 18,446 and vehicle treated animals was required to be greater than 1 and the fold change between the two groups was at least 1.5.

The DAVID Bioinformatic Database was used to analyze the WIN 18,446 upregulated and downregulated gene lists [33], using the Mus musculus genome as a reference. The “Gene

Functional Classification” tool was used to determine which biological functions were the most affected by WIN 18,446 treatment. The biological functions that had an enrichment score of at least 1 were included in the results.

Results

ALDH proteins are expressed in different cell types in both neonatal and adult mouse testes

There is very little data regarding ALDH protein expression in either neonatal or adult mouse testes. To rectify this, immunohistochemical assays were performed to determine the cellular localization pattern of these RA synthesizing enzymes within the testis (Figures 1 and 2).

ALDH1A1 localized primarily to Sertoli cells throughout neonatal testis development (Figure 1

A, E, I, M, Q, and U). ALDH1A2 immunohistochemistry resulted in immunopositive gonocytes at 0 dpp, but no immunopositive cells at 5 dpp. From 10 dpp onwards spermatocytes and, later, spermatids were immunopositive for ALDH1A2 (Figure 1 B, F, J, N, R, and V). ALDH1A3 appeared to be diffusely localized to Sertoli cells up to 5 dpp but shifted to spermatocytes and spermatids by 30 dpp (Figure 1 C, G, K, O, S, and W). ALDH8A1, a previously uninvestigated

RA synthesis enzyme, was present but only within Sertoli cells, though not until 30 dpp (Figure

1 D, H, L, P, T, and X).

The analysis of ALDH enzymes was extended by investigating their localization in the adult testis. Similar to the juvenile testis, both ALDH1A1 and ALDH8A1 were detected predominantly in Sertoli cells (Figure 2 A and D). ALDH1A2 and ALDH1A3 were present in spermatocytes and round spermatids, while both interstitial cells and elongated spermatids were immunopositive for all four ALDH enzymes. Interestingly, ALDH8A1 was the only ALDH enzyme that appeared to be present in spermatogonia, although some spermatogonia were not

ALDH8A1-negative (Figure 2 D, green arrow and asterisks, respectively). Western blots were performed with the ALDH antibodies to ensure specificity (Supplemental Figure 1).

Expression of ALDH enzymes does not cycle in the same manner as RA.

Stage-specific expression of the ALDH enzymes was not visualized via immunohistochemistry as this assay only provides qualitative data. To obtain quantitative information regarding the expression of the ALDH enzymes during the first wave of spermatogenesis and across the cycle of the seminiferous epithelium, spermatogenesis was synchronized using the WIN 18,446/RA treatment protocol [22]. Samples from both synchronized and unsynchronized vehicle control treated animals were assayed for ALDH1A1,

ALDH1A2, and ALDH1A3 protein levels (Figure 3A). Using a novel tandem HPLC MS/MS [8] on synchronized testes during the first wave of spermatogenesis in juvenile mice, we found that the enzyme present at the highest level was ALDH1A1 (Figure 3B). ALDH1A1 levels fell from

600 to 200 pmol/mg of S10 protein from 0-16 days following the neonatal synchrony protocol, with no statistical differences between synchronized and unsynchronized samples. Conversely, the highest levels of ALDH1A2 protein, approximately 15-17 pmol/mg of S10 protein, were observed later during development: 14 days after synchrony treatment in the juvenile testis

(Figure 3C). There was no statistical significance between the levels of ALDH1A2 protein in synchronized and unsynchronized samples except at 4 and 12 days post treatment, where

ALDH1A2 levels were observed to be lower than controls. In both the synchronized and unsynchronized samples, ALDH1A3 concentrations were below 1.3 pmol/mg testiscular S10 protein (data not shown).

This analysis was extended to synchronized adult spermatogenesis to determine if stage- specific cycling of ALDH enzyme levels was occurring. Overall, ALDH1A1 and ALDH1A2 levels varied from ~80-180 pmol/mg and ~10-20 pmol/mg of S10 protein, respectively (Figure

3D and E). ALDH1A3 concentrations were, again, below 1.3 pmol/mg S10 protein (data not shown). To determine whether ALDH protein levels varied significantly across the

spermatogenic cycle, samples were binned into three categories for further analysis. In creating categories, we were interested in assessing ALDH levels before, during, and after the RA peak described previously [7]. If the ALDH enzymes are responsible for testicular RA pulsatility, the enzymes should be most abundant prior to the peak of RA and least abundant just after the peak.

We therefore established our three categories as Stages II-VII (before), Stages VII-IX (during), and Stages X-I (after). In the case of both ALDH1A1 and ALDH1A2, there was no significant difference in ALDH protein levels either before, during, or after the RA peak (Figure 3).

ALDH activity does not undergo cyclic changes

Neither immunohistochemistry nor protein quantification displayed stage-specific expression of ALDH enzymes, yet it is known that RA is present in a stage-specific manner [7].

To further investigate whether the ALDH enzymes are responsible for the stage-specific availability of RA, total ALDH activity across the spermatogenic cycle was determined using

HPLC MS/MS (Figure 4). Samples were again binned and compared as described for the ALDH enzyme levels and showed no changes in ALDH activity before, during, and after the pulse of

RA.

ALDH inhibition across one spermatogenic cycle significantly lowers testicular RA levels

In addition to characterizing the ALDH enzymes, we also investigated the effects of

ALDH inhibition on various aspects of spermatogenesis, as a follow up to previous studies [22,

34]. To better understand the effects of WIN 18,446 on testicular RA levels, animals were treated for 8 days with 125 mg/kg/day of WIN 18,446. Animals were sacrificed at various time points within a 24 hour window following the first and eighth dose. HPLC-MS/MS measurements following a single dose of WIN 18,446 showed a 63% reduction in testicular RA concentrations (Figure 5A). Eight days of WIN 18,446 treatment also resulted in lowered

testicular RA levels to an average of 20% of control (Figure 5B), consistent with previous studies

[8, 22, 34].

WIN 18,446 treatment changes testicular gene expression

To gain a better understanding of the effects of lowered RA levels on the adult murine spermatogenesis, RNA sequencing analysis was performed on the testis of adult animals 24 hours after completion of 8 days of WIN 18,446 treatment. There were 77 and 1218 genes that were shown to be increased or decreased by at least 1.5 fold, respectively (Supplemental Table 1 and 2). A clustering analysis was performed on these gene lists using the DAVID Bioinformatic

Database (Table 1 and Table 2) [33]. According to the analysis, genes associated with DNA packaging/histone modification and gamete generation were both up- and downregulated after

WIN 18,446 treatment. Notable biological functions that were downregulated included fertilization/plasma membrane fusion, cytoskeleton, sperm/egg recognition, acrosomal vesicles, and cell motility. Because there is evidence to suggest that RA plays a role in both meiotic initiation and BTB maintenance (reviewed in [1]), lists were collected looking specifically at genes associated with meiosis and tight junctions, which are essential for BTB integrity, with the majority of genes in both lists showing a downward trend (Supplemental Tables 3 and 4). We also examined the expression of genes involved in vitamin A metabolism and signaling

(Supplemental Table 5). Notably, there were four genes that were downregulated in the vitamin

A metabolism and signaling pathway, including Stra8. There were, however, no significant changes in genes associated with steroidogenesis (Supplemental Table 6).

The BTB is significantly altered in mice treated with WIN 18,446

To determine the effects of this lowered testicular RA environment on the BTB, we performed a biotin-permeability assay on mice treated for 8 days with either WIN 18,446 or

vehicle control. This functional assay allows for the detection of tubules with a disrupted BTB by probing for a biotin tracer molecule that was injected into freshly dissected testes (Figure 6A,

B). There was a small but statistically significant increase in permeable tubules in animals treated with WIN 18,446 (4.8%) compared to controls (2.7%) (Figure 6C).

WIN 18,446 treatment affects meiotic recombination and the frequency of synaptic defects

Because RA has been implicated in playing a role during meiotic initiation [11-14, 35,

36], we investigated whether meiosis was altered in animals treated with WIN 18,446. Animals were treated for 12 days with WIN 18,446 to ensure that spermatocytes analyzed at pachytene had initiated meiosis in an RA-deficient environment. Meiotic preparations were immunostained for SYCP3 and MLH1, markers for the SC and sites of recombination, respectively (Figure 7A and B). The number of MLH1 foci in pachytene spermatocytes was quantified in WIN 18,446 treated animals (26.22 ± 0.26 MLH1 foci/cell) and controls (25.39 ± 0.28 MLH1 foci/cell; Figure

7C). A statistically significant increase in MLH1 foci was detected in WIN 18,446 treated animals compared to controls. To extend our recombination analysis, the distribution of foci across the genome was assessed by analyzing the number of “recombination-less” chromosomes; no significant difference was detected between WIN 18,446 treated and control animals (Figure

7C).

In addition to aberrant recombination, meiotic defects can also cause a cell to undergo apoptosis, thereby adversely affecting fertility [37, 38]. Thus, to determine if lowered testicular

RA levels increased the frequency of meiotic defects, we quantified these defects in pachytene cells immunostained for SYCP1 and SYCP3. Defects were separated into three categories: associations, minor defects, and major defects, and representative images of normal and abnormal cells are shown in Figure 8A-D. Our analysis revealed a significant increase in all

three classes of defects in spermatocytes from WIN 18,446 treated animals by comparison to controls and a statistically significant decrease in cells with no synaptic defects (Figure 8E).

Discussion

This report provides the first comprehensive analysis of the ALDH proteins in the murine testis, particularly throughout postnatal development, as well as a thorough investigation into the effects of short-term inhibition of these RA-synthesizing enzymes on various aspects of spermatogenesis. Here we show that the ALDH enzymes are likely not primarily responsible for endogenous RA pulsatility, but the inhibition of these enzymes has adverse effects on BTB permeability and proper meiotic function through reduced intratesticular RA concentrations.

While there have been a multitude of studies regarding the effects of ALDH inhibition via WIN

18,446 treatment on fertility [22, 39-45], it is not yet clear through which mechanisms ALDH inhibition compromises spermatogenesis. Consistent with previous reports [8, 34], we show our treatment regime with WIN 18,446 causes an 80% reduction in testicular RA levels, allowing for a more thorough investigation of how spermatogenesis proceeds in a lowered RA environment.

While spermatogonial differentiation is the best characterized spermatogenic event known to be under RA control [17-25, 46-48], there is evidence that RA is also important in BTB maintenance [10, 15, 49-52]. Several key genes coding for proteins integral to the BTB are misregulated in vitamin A-deficient mice [15]. Additionally, Sertoli cells cultured in the presence of RA display increased expression of transcripts coding for proteins vital for BTB integrity, such as Tjp1 and Cldn11 [51]. Finally, BTB permeability was compromised in animals with testes treated with a Sertoli cell-specific dominant-negative RA receptor lentivirus [10].

The data presented here show that a lowered testicular RA environment induces a mild increase in BTB permeability. While the increase in permeability reached statistical significance, we were surprised that this increase was not more drastic. It is possible that an 80% reduction in RA concentration was not sufficient to completely eliminate proper BTB function.

RA has also been implicated in playing a vital role during meiosis [11-14, 22, 36]. Animals lacking Stra8, a gene known to be stimulated by RA [13, 14], fail to properly undergo meiosis

[11, 12], indicating that this RA-responsive gene is important for meiotic progression. The data presented here shows, for the first time, that germ cells are not only able to initiate meiosis in an

RA-deficient environment but also have an increased recombination rate and an increase in both major and minor meiotic defects. While the biological significance of the increase in meiotic recombination is not yet fully understood, it is known that increases in meiotic defects can be detrimental to viable sperm production [37, 38]. When taken together, this data provide support for the hypothesis that RA plays a role in proper meiotic regulation, but that meiotic initiation can still take place in a lowered testicular RA environment.

The RNA sequencing analysis performed also supports the meiotic and BTB data. Genes associated with meiotic regulation were found to be significantly downregulated using DAVID

(Table 2) and when lists of genes with known roles in meiosis and BTB integrity were assessed, the majority displayed a downward trend in expression following WIN 18,446 treatment.

Because meiosis and the BTB are associated with a minority of the cells in the adult testis, spermatocytes and Sertoli cells respectively, the gene expression changes detected in this study are likely biologically relevant as molecular signals from these cells will be attenuated by the more abundant cell types when assessing whole testes. A downregulation of genes associated with acrosomal vesicles, cytoskeletal and centriole organization, sperm/egg recognition, and flagellar motion (Table 2) was also detected by RNA sequencing analysis. All of these terms can be associated with spermiogenesis or spermiation, supportive of a role for RA in regulating these processes. This agrees with previously published data suggesting a role for RA during post- meiotic differentiation of germ cells [10, 16, 20, 24, 53-56]. We were not able to detect any

morphological defects associated with spermiation (data not shown), but it is possible that the relatively short treatment window was not sufficient to elicit a visible phenotype.

While there have been multiple studies investigating the effects of WIN 18,446 on spermatogenesis [22, 39-45], less data has been published regarding the regulation of this molecule’s target enzymes: the ALDHs. Recent transcript localization studies have led to the hypothesis that ALDH enzyme activity varies across the spermatogenic cycle, driving the RA pulse [4, 5, 57]. This localization data, however, is incomplete and contradictory, focusing primarily on transcript localization in the adult murine testis. Here we present, for the first time, a comprehensive localization of four ALDH enzymes in the postnatal testis. Our findings show that ALDH1A1 is present in the Sertoli cells and Leydig cells (Figure 1 and 2), agreeing with the transcript localization observed by Vernet et al. 2006 [4] and the protein localization in the human testis [6]. ALDH8A1, known to be present in the human testis and capable of synthesizing RA [58], was similarly localized to both Sertoli and Leydig cells (Figure 1 and 2).

Almost no information is known about the activity of ALDH8A1 in vivo and future studies are required to determine the contribution of this enzyme to testicular RA synthesis, especially in murine models where the activity of the more well-characterized isozymes has been perturbed

[24]. ALDH1A2 localized predominately to the meiotic and post-meiotic germ cells in the adult testis (Figure 2), however it was not expressed in a stage-specific manner, as has been previously reported [4, 5, 59]. Vernet et al. [4] reported Aldh1a3 transcript localization in the Leydig cells, which we were able to confirm at the protein level (Figure 2). In the adult testis, we also were able to detect ALDH1A3 protein in late spermatocytes and round spermatids (Figure 2).

Importantly, the localization patterns of the two enzymes predicted to contribute the most to

testicular RA synthesis in both the mouse and the human, ALDH1A1 and ALDH1A2, were conserved between these two species [6, 8].

To determine if the ALDH enzymes play a role in RA cyclicity, ALDH immunohistochemistry, quantification, and activity were assessed in both the developing and adult testis. ALDH1A1 protein levels drop throughout juvenile testis development and do not vary significantly across the cycle (Figure 3), matching the immunohistochemical Sertoli cell localization data for this enzyme perfectly (Figure 1). As the numbers of germ cells within the testis increase during development, the relative abundance of the Sertoli cells, where ALDH1A1 is predominantly localized, drops. Immunohistochemical analysis of ALDH1A1 in the adult also demonstrated that this isozyme is present in every tubule (Figure 2), consistent with the protein quantification data (Figure 3). While it is known that RA synthesis within Sertoli cells is necessary for spermatogonial differentiation [24], Aldh1a1-null mice are both viable and fertile

[60], indicating that ALDH1A1 is not required for this process.

While ALDH1A2 protein levels did increase throughout juvenile testis development, they remained constant across adult spermatogenesis. Due to the synchrony protocol utilized for protein quantification, the testes of the synchronized animals were developmentally delayed by approximately seven days compared to controls [22, 61]. Therefore the significant differences in observed in ALDH1A2 protein level between controls and synchronized developing testis are likely the result of new, ALDH1A2-expressing cell types entering the testicular milieu in the unsynchronized controls. Specifically, the observed statistically significant differences correspond nicely with the appearance of preleptotene spermatocytes and round spermatids in the unsynchronized controls, cell types that are not present in the corresponding synchronized animals, at 4 and 12 days post treatment, respectively. It is not likely that these increases were

responsible for RA pulsatility, as the drastic changes seen in RA levels in synchronized neonatal animals [7] were not observed in ALDH1A2 levels. As was observed with ALDH1A1,

ALDH1A2 protein levels did not change significantly in a stage-specific manner in the adult testis, matching the corresponding immunohistochemical data (Figure 3). Since ALDH1A3 levels were not detected via HPLC-MS/MS analysis, we were not able to detect stage specificity when analyzing protein localization. However, based on the activity of ALDH1A3 and its low expression levels, this enzyme is not expected to provide a significant contribution to testicular

RA synthesis in either the human or mouse [6, 8], and is unlikely to contribute to cyclic RA concentrations.

In addition to localization and quantification, stage-specific ALDH enzyme activity during adult spermatogenesis was not detected in our assay. However, despite multiple approaches, our techniques still have limitations and therefore we cannot definitively rule out

ALDH as contributing to the RA pulse. First, quantitative data from immunohistochemical analysis is not possible. Second, mass spectrometry protein and enzyme activity assays were performed on the S10 fraction of whole testes, not within the seminiferous tubules themselves.

While our assays suggest that ALDH availability and activity are not responsible for synthesizing RA in a pulsatile manner along testis tubules, in vivo it is possible that the presence of ALDH substrate or cofactors are responsible for RA pulse regulation. Specifically retinaldehyde could be regulated in a stage-specific manner, as Rdh11, Dhrs4, and Lrat, genes known to encode enzymes responsible for retinaldehyde availability, vary across the cycle [7]. It is also possible that the retinoid binding proteins contribute to the availability of RA across the cycle, as Crabp1, a known binder of RA, displays a stage-specific mRNA expression pattern based on microarray data [7].

It is clear that RA acts as more than just an on/off switch for spermatogonial differentiation. Here we provide evidence that a lowered testicular RA environment compromises BTB integrity as well as increases the number of meiotic defects, both of which have been shown to have an adverse effect on fertility. It will be important for future studies to determine if these spermatogenic defects are present in men with lowered testicular RA levels and if these defects can be rescued via pharmaceutical intervention.

ACKNOWLEDGEMENTS

The authors would like to thank Mark Wildung for his support and expertise with RNA sequencing, Dr. Mary Zoulas of the Seattle Animal Shelter for kindly providing dog testicular tissue for controls, and Dr. John Amory for giving us WIN 18,446.

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FIGURES

Figure 1. ALDH enzymes are differentially localized in the neonatal murine testis.

Images are representative murine testicular cross-sections displaying immunohistochemical analysis of ALDH localization at various neonatal ages. ALDH1A1 is represented in A, E, I, M,

Q, and U for 0, 5, 10, 15, 20, and 30 dpp, respectively. ALDH1A2 is represented in B, F, J, N,

R, and V while ALDH1A3 is presented in C, G, K, O, S, and W for the same time points.

Finally, ALDH8A1 is displayed in D, H, L, P, T, and X. Negative controls are shown in Y-BB.

Brown staining indicates an immunopositive reaction. Arrows indicate immunopositive cells while the respective colors indicate the following cell types: Red – Sertoli cells, Yellow –

Leydig/Interstitial cells, Dark Blue – Spermatid, Light Blue – Spermatocyte, Green –

Gonocyte/Spermatogonia. Scale bars represent 100 μm.

Figure 2. ALDH enzymes locate to different cell types in the adult murine testis.

Images are representative adult murine testicular cross-sections displaying immunohistochemical analysis of ALDH localization. ALDH1A1, 1A2, 1A3, and 8A1 localization can be seen in A,

B, C, and D, respectively. Negative controls for each assay are shown in the picture inserts.

Brown staining indicates an immunopositive reaction. Arrows indicate immunopositive cells while the respective colors indicate the following cell types: Red – Sertoli cells, Yellow –

Leydig/Interstitial cells, Dark Blue – Spermatid, Light Blue – Spermatocyte, Green –

Gonocyte/Spermatogonia. Asterisks (*) represent immunonegative spermatogonia. Scale bars represent 100 μm. A summary of the adult localization data is represented in E. A solid line underneath a cell indicates that cell is immunopositive for the respective ALDH. ALDH1A1,

1A2, 1A3, and 8A1 are represented by blue, green, red, and yellow, respectively. Stage diagram adapted from Hogarth and Griswold, 2010 [62].

Figure 3. ALDH1A1 and ALDH1A2 levels do not vary in a manner similar to RA.

A represents the cell types entering the testicular environment following synchrony protocol (top row) or control (bottom row). ALDH1A1 (B and D) and ALDH1A2 (C and E) protein levels were quantified in both synchronized (red) and control (blue) animals using a tandem HPLC

MS/MS approach. In the neonatal analysis (B and C) the vertical axis represents ALDH protein per mg of S10 protein. The horizontal axis represents time following neonatal synchrony

treatment. Vertical error bars represent the SEM. Student t-tests were used to compare the protein levels in synchronized and control animals (* p < 0.05, ** p < 0.01). In the synchronized adult analysis (D and E), 25 animals were collected at 12 hour intervals between 42 and 50 days post synchrony. The midpoint of synchrony was determined and used to bin each sample into three categories (Stages II-VII, Stages VIII-IX, and Stages X-I). ALDH protein per mg of S10 protein was determined and averaged for both the unsynchronized controls (blue) and the three bins (red). The vertical control error bar is representative of the SEM for each category.

Figure 4. ALDH activity does not undergo cyclic changes

The graph represents mean ALDH activity (y-axis) within each category. Four categories used were: Control (blue), Stages II-VII, Stages VIII-IX, and Stages X-I (red). Error bars are representative of the SEM for each category.

Figure 5. Animals treated with WIN 18,446 have lowered testicular RA levels.

Adult mice were treated daily with WIN 18,446 for either 1 (A) or 8 (B) days (n = 24). Their testes were collected within a 24 hour window of the final treatment. The RA levels were measured (y-axis) and a student t-test was used to determine statistical significance (* p < 0.05,

** p < 0.01).

Figure 6. Blood-testis barrier permeability is adversely affected in animals treated with WIN

18,446.

Adult mice (n = 4) were treated daily with WIN 18,446 for 8 days. BTB integrity was assessed using a biotin permeability assay. Representative images of a tubule with an intact BTB and a compromised BTB are shown in A and B, respectively. Brown staining is indicative of the presence of the biotin tracer. The percentage of permeable tubules was determined for each treatment and a student t-test was used to determine statistical significance (C) (* p < 0.05).

Figure 7. WIN 18,446 treatment increases recombination rate.

Animals (n = 4) were treated with WIN 18,446 for 12 days. Meiotic spreads and immunohistochemistry were performed to determine recombination rates. A and B are representative images of cells from control and WIN 18,446 treated animals, respectively.

Purple and green staining is representative of SYCP3 and MLH1, respectively. e0, e1, e2, and e3 denote the percentage of chromosomes containing 0, 1, 2, and 3 exchanges, represented by

MLH1. A student t-test was used to determine if there was a statistically significant difference in recombination rate between control and WIN 18,446 treated animals (* p < 0.05).

Figure 8. WIN 18,446 induces meiotic defects.

Animals (n = 4) were treated daily with WIN 18,446 for 12 days. Meiotic spreads and immunohistochemistry were performed to assess meiotic defects. Purple and green staining is

representative of SYCP3 and SYPC1, respectively. Cells were binned into four categories 1) cells with no detectable meiotic defects (A), 2) cells containing association of two non- homologous chromosomes at their distal ends (B), 3) cells with minor synaptic defects, such as forks, bubbles and gaps (C), or 4) cells with major synaptic defects, such as partial or complete asynapsis of homologous chromosomes (D). The circles highlight the specific meiotic defects in each cell with red representative of associations, blue representative of minor defects, and green representative of major defects. E shows the percentage of cells containing each defect. The values do not add up to 100% because some cells, such as the one depicted in D, displayed two different types of defects.

Supplemental Figure 1: ALDH Western Blots

Western Blots were performed on protein isolated from adult mouse testis. Bands were detected in all blots at approximately 58 kDA. The expected sizes of ALDH1A1, ALDH 1A2,

ALDH1A3, and ALDH8A1 are 54.5, 56.7, 56.2, and 53.7 kDA, respectively. Ponceau staining was used to ensure equal loading.

TABLES

Table 1. Biological functions upregulated after 8 days WIN 18,446 treatment.

WIN 18,446 Upreguated DNA Packaging/Histone modification 5.57 Endoplasmic reticulum 1.55 Sexual Reproduction/Gamete generation 1.17

List of biological functions that are upregulated after WIN 18,446 are listed in the left column.

The right column denotes the gene enrichment score as reported by DAVID Bioinformatic

Database [33].

Table 2. Biological functions downregulated after 8 days WIN 18,446 treatment.

WIN 18,446 Downregulated Spermatogenesis/Gamete generation 8.77 Organelle envelopes and membranes 4.24 Translation/Ribosomes 3.69 DNA packaging/Histone modification 3.61 DnaJ/Heatshock proteins 3.33 Fertilization/Plasma membrane fusion 3.20 Cytoskeleton/Non-membrane bound 2.88 organelles Germ cell and spermatid development 2.78 Vesicles (Acrosomal, secretory, cytoplasmic 2.40 Ion transmembrane transporters 2.30 Chromosome and DNA condensation 1.83 RNA splicing and processing 1.77 Flagella 1.64 Cell motility 1.63 Proteinacious extracellular matrix 1.53 Sperm-egg recognition 1.50 Viral nucleoprotein 1.43 Hyaluronglucosaminidase activity 1.33 Meiosis/Male germ cell nucleus 1.28 Microtubule associated complex 1.27 Enzyme inhibitor activity 1.26 ETC/ATP synthase/Ion transport 1.25 Lipocalin/Calycin 1.24 Microtubule 1.06 organization/Centrosome/Centriole

List of biological functions that are downregulated after WIN 18,446 are listed in the left column. The right column denotes the gene enrichment score as reported by DAVID

Bioinformatic Database [33].

Supplemental Table 1: Genes upregulated after 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Lars2 3033.426 25.17919 133.2929 117.6193 125.4561 4974.867 1342.897 3158.882 Gm15564 11.48413 15.65332 0.454042 1.113402 0.783722 21.53819 2.997514 12.26785 Nt5c1a 1.254165 8.019027 0.076387 0.280974 0.178681 1.424848 1.440843 1.432845 Paqr5 2.663468 7.768599 0.353536 0.433471 0.393504 0.378995 5.734947 3.056971 Uck2_1 2.649679 3.644215 2.004132 0 1.002066 2.578145 4.725344 3.651745 Gm21983 1.537404 3.342006 0.772006 0.54089 0.656448 0.851246 3.536459 2.193852 Gm10076 1.695158 3.243758 1.213439 0.29756 0.7555 4.214664 0.686652 2.450658 Gm15017 1.167088 3.124158 0.73728 0.361591 0.549436 2.181429 1.251618 1.716524 Scara3 2.446525 3.035797 1.094768 1.308738 1.201753 3.837689 3.458868 3.648279 Hist1h2ao 1.710374 2.649988 1.359052 0.714143 1.036598 2.747336 2.746606 2.746971 Gm9800 1.505583 2.543744 1.32549 0.625071 0.97528 2.557695 2.404032 2.480863 Cox7a1 1.355457 2.533599 0.512045 1.255636 0.883841 2.305457 2.173139 2.239298 Hmga1 12.59336 2.52694 9.834347 6.660552 8.247449 24.36833 17.31328 20.84081 Gm26917 259.7916 2.503033 166.8665 178.8234 172.845 378.4823 486.7909 432.6366 Gm14089 1.632985 2.451426 1.172228 1.077952 1.12509 3.581433 1.934716 2.758075 Mrm1 1.307866 2.347314 1.104788 0.836653 0.970721 2.779732 1.777441 2.278586 1700012C14Rik 1.332837 2.300726 1.498266 0.551107 1.024687 2.31287 2.402176 2.357523 Hist1h2ac 2.855979 2.297416 1.762036 2.64053 2.201283 3.651921 6.462603 5.057262 Hist2h2ab 1.208282 2.218061 1.469246 0.514698 0.991972 2.025064 2.375443 2.200253 Nop56 13.40275 2.160579 11.89058 11.20607 11.54833 16.17363 33.72851 24.95107 Insl3 87.66026 2.055786 97.82932 68.22761 83.02847 78.1265 263.251 170.6887 Hist1h2bj 12.42532 2.007324 9.862668 14.80729 12.33498 35.47317 14.04744 24.7603 Fam132a 1.632053 1.997305 1.432676 1.840252 1.636464 1.711371 4.825662 3.268517 4933406F09Rik 3.43374 1.982501 3.853324 3.136473 3.494898 3.290764 10.56651 6.928638 Arv1 1.875524 1.957485 1.759852 2.157755 1.958803 3.292945 4.375709 3.834327 Snhg12 32.17499 1.949423 45.22054 22.55744 33.88899 49.80411 82.32386 66.06399 1110038B12Rik 8.219954 1.92634 8.850938 8.896229 8.873584 10.98879 23.19828 17.09354 Mgp 2.071952 1.901778 2.493435 2.101826 2.297631 3.007118 5.732048 4.369583 Rpl13 2.018063 1.895455 1.981126 2.526221 2.253673 4.9187 3.624773 4.271737 Scand1 5.447496 1.882118 6.630271 5.720673 6.175472 7.265683 15.98025 11.62297 Ifitm2 1.329422 1.878771 1.932214 1.093425 1.512819 3.441639 2.242843 2.842241 2810008D09Rik 6.639031 1.872558 6.593784 8.623616 7.6087 8.906459 19.589 14.24773 Hist2h2ac 1.814717 1.838048 1.25451 3.076309 2.165409 3.22764 4.732614 3.980127 Wfdc10 4.821651 1.808472 6.700089 5.227715 5.963902 11.23094 10.34016 10.78555 RP23-12N20.2 2.343592 1.783555 2.900171 3.08178 2.990975 6.839841 3.829293 5.334567 Trip6 1.046181 1.782149 1.376702 1.298442 1.337572 2.520284 2.247223 2.383753 Snhg1 19.29037 1.748234 21.62994 29.93246 25.7812 30.73369 59.40945 45.07157 Prnp 2.46273 1.746336 3.38085 3.218674 3.299762 7.061016 4.463968 5.762492

Ntn4 1.235603 1.730538 1.519573 1.863148 1.69136 2.611742 3.242185 2.926963 Hist1h2bp 11.38118 1.714182 12.93602 18.93592 15.93597 39.81975 14.81456 27.31715 1500012F01Rik 1.486162 1.713926 2.290828 1.872523 2.081675 2.84463 4.291045 3.567837 Hist1h4k 1.997992 1.702827 2.389542 3.296046 2.842794 4.610914 5.070657 4.840786 Serping1 1.970296 1.686947 2.88595 2.85043 2.86819 4.460192 5.21678 4.838486 Gas5 11.67514 1.686603 17.32482 16.6836 17.00421 19.2101 38.14861 28.67935 AB124611 1.008168 1.676253 1.481285 1.500342 1.490814 2.568346 2.429617 2.498982 Gadd45gip1 1.201694 1.674019 2.466192 1.099563 1.782877 2.163099 3.806043 2.984571 Tmem35 3.246262 1.673679 4.275761 5.361659 4.81871 6.781746 9.348199 8.064972 Frmpd1 1.124828 1.651038 1.802928 1.652563 1.727746 2.634561 3.070586 2.852573 Hba-a2 4.03283 1.646227 7.68451 4.796644 6.240577 9.346261 11.20055 10.27341 Ndrg4 2.137301 1.641922 3.432492 3.226577 3.329534 5.286464 5.647207 5.466835 Gpr56 3.935603 1.640085 5.643624 6.653508 6.148566 10.4557 9.712636 10.08417 Tns3 1.25869 1.63983 1.94185 1.992601 1.967226 3.20562 3.246212 3.225916 Cmtm3 1.449592 1.625557 2.018394 2.616168 2.317281 3.672182 3.861565 3.766874 Rbm42 2.21065 1.625348 3.527108 3.543036 3.535072 6.641987 4.849457 5.745722 Rab3b 1.075286 1.616767 1.930484 1.556362 1.743423 2.993692 2.643725 2.818709 Hist1h2ba 126.9038 1.603576 230.0681 190.4382 210.2532 435.3591 238.9547 337.1569 Ptgis 1.142105 1.600819 1.887804 1.914026 1.900915 3.175729 2.910311 3.04302 Gm28045 1.217613 1.581249 1.714514 2.475131 2.094823 1.565248 5.059623 3.312436 Mfge8 8.343569 1.58076 12.27944 16.45386 14.36665 24.20242 21.21802 22.71022 Slc7a4 2.578403 1.580331 4.146559 4.739412 4.442985 6.795824 7.246953 7.021389 Srl 1.087421 1.5732 1.820091 1.97412 1.897105 3.16313 2.805922 2.984526 Aqp8 7.834537 1.569164 13.47356 14.05644 13.765 21.74176 21.45732 21.59954 Serpina5 15.14545 1.565996 24.3972 29.12066 26.75893 39.00936 44.79941 41.90438 Anxa6 1.274906 1.562831 2.385464 2.144867 2.265166 3.150529 3.929615 3.540072 Hist1h2bm 4.084187 1.53855 5.870371 9.296982 7.583677 18.72201 4.613722 11.66786 Gsta2 2.00172 1.532724 4.11724 3.397793 3.757516 6.36596 5.152512 5.759236 Rhox5 2.909998 1.530377 6.427045 4.54627 5.486657 8.983333 7.809978 8.396655 Ppp1r14a 1.091804 1.529737 1.755031 2.367028 2.06103 3.160777 3.14489 3.152834 Olfml2b 1.736136 1.528997 3.462713 3.101158 3.281936 5.151709 4.884433 5.018071 Prnd 4.75231 1.526881 8.822925 9.216473 9.019699 15.36349 12.18053 13.77201 Fam210b 1.664083 1.522765 2.859919 3.506548 3.183234 5.004824 4.689809 4.847317 Hist1h2bl 1.493419 1.515711 4.891976 0.899708 2.895842 7.394405 1.384116 4.389261 Ugt1a7c 1.877617 1.513344 2.981972 4.33327 3.657621 7.17484 3.895637 5.535238 Ltbp1 1.136981 1.510054 1.885185 2.573096 2.22914 3.260198 3.472046 3.366122 4930591A17Rik 1.041672 1.507152 2.402511 1.705417 2.053964 3.090626 3.100646 3.095636 Speer7-ps1 2.795353 1.506375 5.76535 5.275296 5.520323 6.973848 9.657503 8.315676

Supplemental Table 2: Genes downregulated after 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Ighv1-84 -3.17802 -38.2189 4.248075 2.278748 3.263411 0.170775 0 0.085387 Gm20896 -1.24175 -27.0242 1.21423 1.364704 1.289467 0 0.09543 0.047715 RP24-554B1.5 -1.63712 -20.8466 3.334398 0.104828 1.719613 0.164978 0 0.082489 Mup12 -2.69021 -17.6236 5.333892 0.370182 2.852037 0.32366 0 0.16183 Mup7 -1.45348 -15.4526 2.886882 0.221225 1.554053 0.116054 0.085084 0.100569 Mup17 -1.36726 -15.0965 2.312211 0.616305 1.464258 0.193987 0 0.096993 Gm14260 -2.76472 -12.9921 0.716863 5.273673 2.995268 0.461091 0 0.230546 1700063H06Rik -2.29099 -9.50401 4.607252 0.513541 2.560397 0.538803 0 0.269402 RP23-110G23.2 -1.03333 -9.44143 1.038353 1.273124 1.155738 0 0.244823 0.122411 Gm16271 -1.59043 -8.96195 2.541604 1.038754 1.790179 0 0.399506 0.199753 Gm7568 -1.50831 -8.88841 2.601575 0.797448 1.699512 0.119525 0.262886 0.191205 Mup9 -1.69503 -8.3936 3.543664 0.304904 1.924284 0.399879 0.058633 0.229256 Mup3 -2.18672 -7.83602 4.799221 0.213975 2.506598 0.392878 0.246886 0.319882 Gm20481 -1.08452 -6.8878 1.321874 1.215563 1.268719 0.21256 0.155836 0.184198 Hist1h2ah -1.71592 -6.79684 0.920416 3.103437 2.011927 0.592019 0 0.296009 Eif3j2 -1.97369 -6.78921 2.467888 2.161341 2.314615 0.20157 0.480281 0.340925 Gm7094 -1.14534 -6.6712 0.509247 2.185356 1.347301 0.163776 0.24014 0.201958 Amd2 -1.89431 -6.24205 1.834147 2.6772 2.255674 0.393246 0.32949 0.361368 Gm20656 -3.69194 -6.17454 3.957968 4.852865 4.405416 0.57859 0.848373 0.713481 Gm8362 -1.77739 -5.93631 3.584313 0.6906 2.137457 0.526962 0.193168 0.360065 Gm14478 -1.32142 -5.78102 1.758342 1.437269 1.597806 0 0.552776 0.276388 Mup10 -1.91715 -5.68305 4.488766 0.164289 2.326528 0.81876 0 0.40938 Gm12665 -2.18653 -5.61294 5.154733 0.166322 2.660527 0.436258 0.51174 0.473999 Samt1 -2.64855 -5.5458 2.746516 3.715867 3.231192 0.18275 0.982526 0.582638 1700073E17Rik -4.08392 -5.47396 7.735316 2.25816 4.996738 0.852928 0.972711 0.91282 Rps10-ps1 -1.96956 -5.42318 2.994125 1.835549 2.414837 0.361096 0.529466 0.445281 RP23-413D14.1 -1.42828 -5.39398 2.044435 1.462231 1.753333 0.328749 0.321358 0.325053 Gm5797 -7.2611 -5.27301 12.99269 4.92809 8.960388 2.164401 1.234182 1.699292 Prss23os -1.71007 -5.26692 2.511166 1.710523 2.110844 0.5384 0.263148 0.400774 Apoa1 -2.68838 -5.26418 5.80566 0.832012 3.318836 1.260913 0 0.630457 Arg1 -1.06097 -5.21412 2.083564 0.541897 1.312731 0.324888 0.178641 0.251765 Gm14122 -3.73739 -5.12798 4.118461 5.167079 4.64277 0.184816 1.625946 0.905381 Gm12279 -1.41071 -4.9923 1.5849 1.943247 1.764074 0.407768 0.29895 0.353359 Klk1b3 -1.13012 -4.98674 1.154407 1.672768 1.413588 0.270008 0.29693 0.283469 Gc -1.37704 -4.87834 3.085833 0.378354 1.732093 0.66161 0.048505 0.355058 Gm9843 -2.4342 -4.61246 2.588671 3.627394 3.108032 0.475729 0.871938 0.673834 Gm15389 -1.34864 -4.5682 2.938491 0.514698 1.726594 0.360011 0.395907 0.377959 CH36-116L8.4 -1.30191 -4.48098 2.379374 0.97245 1.675912 0 0.748012 0.374006

Aldob -1.04078 -4.4234 2.259676 0.429919 1.344797 0.35083 0.257207 0.304019 Gm3127 -1.40565 -4.39704 3.012787 0.626098 1.819443 0.394138 0.433437 0.413788 1700046C09Rik -1.83063 -4.36201 4.11899 0.631287 2.375138 0.441561 0.64745 0.544506 Gm21815 -2.37037 -4.32424 3.929789 2.237075 3.083432 0.631917 0.794199 0.713058 Gm4202 -5.5651 -4.31597 7.82915 6.657595 7.243373 0.974665 2.381881 1.678273 Slfn5os -6.5254 -4.24372 14.2873 2.7869 8.537099 1.879711 2.143693 2.011702 Igkv14-100 -1.08487 -4.22159 1.863843 0.979396 1.42162 0.171263 0.502237 0.33675 CH36-396A5.1 -1.01854 -4.13579 1.708902 0.9778 1.343351 0.219836 0.429787 0.324811 4930449I24Rik -5.62335 -4.06502 9.900131 5.015931 7.458031 1.894563 1.774804 1.834684 Gm16726 -2.86688 -4.03333 4.805873 2.818144 3.812008 0.806393 1.083863 0.945128 Gm7950 -1.04816 -4.0316 2.787799 0 1.3939 0.128081 0.563406 0.345744 Alb -10.9007 -3.89064 25.31652 4.026887 14.6717 6.337468 1.204581 3.771024 Gm10184 -3.76461 -3.88867 6.776737 3.358941 5.067839 0.340052 2.26641 1.303231 Gm15669 -1.2866 -3.87094 1.948885 1.520609 1.734747 0.227916 0.668376 0.448146 RP23-402F16.1 -2.72004 -3.82246 6.562827 0.804668 3.683748 0.844252 1.083169 0.96371 RP24-120M11.1 -1.07494 -3.80905 2.420575 0.494645 1.45761 0.194617 0.570723 0.38267 Tmem132cos -3.04391 -3.74818 7.396202 0.906849 4.151525 1.01942 1.195801 1.107611 Ndufs5 -2.26089 -3.72611 4.984696 1.195775 3.090235 1.045498 0.613196 0.829347 Gm6660 -2.76788 -3.65746 5.177341 2.441515 3.809428 0.768486 1.314615 1.04155 Gm9830 -1.42006 -3.63788 2.329804 1.586985 1.958394 0.832526 0.244143 0.538334 4930562F07Rik -3.40463 -3.52264 6.96084 2.547668 4.754254 0.935548 1.763707 1.349627 Gm15522 -1.09108 -3.50707 1.305877 1.746694 1.526286 0.534514 0.335891 0.435202 Smim4 -1.1942 -3.49183 1.416772 1.930117 1.673445 0.810025 0.148465 0.479245 Gm14590 -2.46849 -3.47571 4.165012 2.766141 3.465577 1.339483 0.654685 0.997084 Gm16404 -1.40452 -3.47395 1.991285 1.953212 1.972249 0.384243 0.751209 0.567726 RP24-162O23.1 -2.99643 -3.46091 5.718904 2.709163 4.214033 1.086812 1.348404 1.217608 Gm11749 -1.37984 -3.4439 2.025916 1.862982 1.944449 0.651542 0.477671 0.564606 Gm11269 -1.63087 -3.44098 3.44576 1.152231 2.298996 0.302228 1.034016 0.668122 Gm4181 -1.92585 -3.38476 5.085095 0.381725 2.73341 0.734255 0.880871 0.807563 Fabp1 -1.71372 -3.34811 4.887111 0 2.443555 1.300728 0.158936 0.729832 Sap25 -1.62175 -3.33457 2.744884 1.887965 2.316424 0.947357 0.441983 0.69467 1810009A15Rik -2.53089 -3.32438 3.678637 3.560825 3.619731 1.203819 0.973866 1.088842 Gm3543 -1.01695 -3.31256 1.649419 1.26397 1.456694 0.198922 0.680575 0.439749 Gm14595 -2.03987 -3.30374 3.297301 2.553361 2.925331 1.116236 0.654685 0.88546 Gm8730 -1.00599 -3.20942 1.647117 1.275493 1.461305 0.501839 0.408797 0.455318 Ube4bos3 -1.16083 -3.18536 3.081751 0.302283 1.692017 0.713594 0.348775 0.531185 Hist1h3c -2.4755 -3.14866 3.049232 4.205997 3.627614 1.225805 1.078422 1.152114 Gm12901 -1.30186 -3.14015 2.102078 1.718239 1.910159 0.225345 0.991256 0.608301 Gm9805 -12.6899 -3.12144 23.97927 13.36408 18.67167 6.309672 5.653832 5.981752 4933400A11Rik -2.58486 -3.10092 6.277191 1.353225 3.815208 1.419793 1.040905 1.230349 Appbp2os_2 -1.20207 -3.05411 2.494908 1.07965 1.787279 0.755173 0.415235 0.585204 Oaz1-ps -1.28693 -3.03083 0.821076 3.020165 1.920621 0.880204 0.387187 0.633695 Gm5169 -2.83236 -3.02399 5.339133 3.124377 4.231755 0.624395 2.174397 1.399396

Gm17111 -1.80595 -2.99741 4.308153 1.112048 2.7101 1.166752 0.641543 0.904148 Rnf133 -7.75347 -2.96076 15.30504 8.11052 11.70778 7.174672 0.733957 3.954315 1700010K23Rik -5.25934 -2.93328 11.40117 4.558361 7.979764 2.869558 2.571291 2.720425 Gm12319 -3.76947 -2.93004 6.003976 5.441091 5.722533 2.182758 1.723361 1.953059 Gm10230 -2.23273 -2.90377 1.934785 4.876272 3.405529 0.622234 1.723361 1.172797 RP24-85N11.1 -3.56313 -2.90249 5.158851 5.713146 5.435998 1.391508 2.354234 1.872871 1700019G24Rik -7.05156 -2.89473 17.23036 4.316094 10.77323 4.647581 2.79575 3.721666 4930538E20Rik -1.14411 -2.88932 2.513273 0.986088 1.74968 0.452636 0.758502 0.605569 Gm10108 -2.8949 -2.88077 5.275027 3.593173 4.4341 1.696468 1.38194 1.539204 1700011H14Rik -10.6165 -2.87305 22.54292 10.02623 16.28458 5.638802 5.697308 5.668055 Gm14511 -1.23729 -2.85345 0.98674 2.822966 1.904853 1.128318 0.206803 0.667561 Gm10220 -1.33548 -2.8486 2.988401 1.127409 2.057905 0.621006 0.823846 0.722426 4930527E20Rik -7.33189 -2.84281 15.13377 7.487313 11.31054 4.952463 3.004834 3.978649 4930522O17Rik -7.67663 -2.839 15.01082 8.691139 11.85098 4.022945 4.325751 4.174348 Gm10413 -1.66564 -2.82468 3.304686 1.852286 2.578486 0.668045 1.157637 0.912841 Klk1b9 -1.41706 -2.81507 3.213522 1.18203 2.197776 0.55119 1.010246 0.780718 RP23-204E23.2 -2.9676 -2.81377 7.138674 2.068827 4.603751 2.170597 1.101703 1.63615 Gm14525 -1.77087 -2.78285 2.713321 2.814988 2.764154 0.80549 1.181071 0.993281 Gm14594 -1.31357 -2.76544 1.56188 2.553361 2.05762 0.669742 0.818356 0.744049 Speer4c -4.11016 -2.7226 9.834347 3.15802 6.496184 2.710939 2.061105 2.386022 Gm3029 -2.69146 -2.71285 6.569436 1.956163 4.262799 1.992026 1.150643 1.571334 1700057G04Rik -8.39291 -2.70677 18.45903 8.161637 13.31033 3.734981 6.099859 4.91742 RP23-289I3.1 -1.30822 -2.69957 2.923675 1.232247 2.077961 0.763965 0.775512 0.769739 1700034J05Rik -21.6141 -2.69289 40.56818 28.19524 34.38171 13.81434 11.7208 12.76757 Gm21671 -3.0502 -2.68614 7.30237 2.416007 4.859188 2.087528 1.530447 1.808988 Gm14625 -1.95672 -2.66067 3.584313 2.685667 3.13499 1.793133 0.563406 1.17827 Nme8 -25.6025 -2.64971 59.82073 22.42298 41.12186 12.34017 18.69858 15.51937 Gm15350 -1.96357 -2.63541 4.667135 1.661335 3.164235 1.549386 0.851936 1.200661 Serpina1c -1.01918 -2.6297 2.533512 0.755596 1.644554 1.057021 0.193736 0.625378 Gm3173 -1.73984 -2.62388 4.033045 1.589438 2.811241 0.648521 1.49429 1.071405 Guca1a -2.002 -2.61338 4.386772 2.098974 3.242873 1.169933 1.311812 1.240873 1700099I09Rik -1.00865 -2.61288 2.106037 1.161996 1.634016 1.151426 0.099312 0.625369 Gm14781 -1.31793 -2.60779 3.01504 1.260253 2.137646 1.057798 0.581634 0.819716 Hmgb4 -5.35565 -2.60763 9.868873 7.505205 8.687039 2.892638 3.77014 3.331389 Gm21093 -1.49306 -2.60136 2.779415 2.071433 2.425424 1.402149 0.462586 0.932368 Gm26951 -2.85204 -2.59843 1.379489 7.893162 4.636325 2.267538 1.301024 1.784281 Spaca5 -2.66413 -2.59824 6.698185 1.963894 4.33104 1.685865 1.647964 1.666915 Izumo3 -12.4445 -2.59193 28.51017 12.01341 20.26179 6.254805 9.379714 7.81726 BC050972 -2.91252 -2.57307 6.37817 3.149833 4.764001 1.196558 2.506409 1.851484 Ms4a5 -15.102 -2.5644 33.70388 15.80712 24.7555 10.62211 8.684921 9.653516 Capsl -2.96664 -2.5608 5.024101 4.710628 4.867364 2.407813 1.393627 1.90072 Gm10053 -1.24128 -2.55989 2.74961 1.324438 2.037024 0.757958 0.833533 0.795746 Hist1h3h -1.5847 -2.55976 1.587214 3.614155 2.600685 1.604284 0.427695 1.01599

1700080G11Rik -1.48452 -2.55757 2.778665 2.096567 2.437616 1.099851 0.806343 0.953097 Gm7964 -1.75394 -2.55282 2.811832 2.955075 2.883454 1.248789 1.010246 1.129518 0610040B10Rik -1.6799 -2.55225 3.684023 1.840252 2.762138 0.877626 1.286843 1.082235 Gm6647 -2.49875 -2.54778 6.978507 1.247801 4.113154 1.309183 1.919625 1.614404 RP24-364H18.1 -1.92316 -2.53239 4.016903 2.339435 3.178169 0.710518 1.799501 1.255009 RP23-125L8.1 -3.09625 -2.52552 6.242536 4.009225 5.125881 1.816421 2.242843 2.029632 Apoc1 -4.21142 -2.52354 10.75961 3.191702 6.975658 3.237085 2.291396 2.76424 Gm5646 -2.1542 -2.51844 3.785115 3.360674 3.572895 2.098805 0.738583 1.418694 Ahsg -1.21946 -2.50953 3.666991 0.387595 2.027293 1.138652 0.477023 0.807837 Gm12720 -1.36401 -2.49889 2.369292 2.178742 2.274017 0.888969 0.931053 0.910011 Gm1840 -10.3198 -2.4951 17.5436 16.90088 17.22224 6.278414 7.526439 6.902427 Serpina1b -1.18761 -2.49067 3.164288 0.804335 1.984312 1.265853 0.327546 0.796699 Meox1 -1.32194 -2.48015 2.635146 1.794974 2.21506 1.076156 0.710074 0.893115 Gm3618 -3.77962 -2.47955 5.824509 6.843872 6.334191 3.278072 1.831071 2.554571 1700045H11Rik -1.139 -2.46993 2.671613 1.156117 1.913865 0.808659 0.741074 0.774867 1700121N20Rik -1.55844 -2.46196 3.357458 1.891402 2.62443 0.933856 1.19813 1.065993 Apoa2 -2.74727 -2.45934 7.98294 1.276681 4.62981 2.45572 1.309369 1.882544 Ndufa4 -2.72545 -2.44352 6.440108 2.7869 4.613504 1.096498 2.679616 1.888057 Gm7275 -2.62788 -2.44139 6.137655 2.764425 4.45104 1.047372 2.598939 1.823155 Spata45 -22.9547 -2.44048 55.08788 22.6924 38.89014 14.00511 17.86577 15.93544 Fgg -1.17392 -2.43591 2.740945 1.241988 1.991467 1.073128 0.561965 0.817547 Speer5-ps1 -5.70941 -2.43261 11.15427 8.235165 9.694719 4.011555 3.959072 3.985313 Rsph3a -37.8477 -2.4308 64.02969 64.56977 64.29973 24.31631 28.58779 26.45205 Gm9780 -1.03186 -2.42572 1.871865 1.639353 1.755609 0.816998 0.630498 0.723748 Gm1993 -1.22342 -2.41933 2.554338 1.616451 2.085395 0.635988 1.087957 0.861973 Nmur1 -1.60204 -2.41729 3.736145 1.728637 2.732391 0.99752 1.26319 1.130355 4921507G05Rik -5.31123 -2.41211 9.020413 9.124443 9.072428 4.278973 3.243421 3.761197 4930438E09Rik -1.10021 -2.40577 2.882231 0.883476 1.882854 0.772447 0.792835 0.782641 Csad -1.781 -2.4032 4.032155 2.068335 3.050245 1.296756 1.241733 1.269244 4932411N23Rik -1.14566 -2.39558 1.887367 2.045799 1.966583 0.738937 0.902905 0.820921 Oxct2a -2.76793 -2.38866 2.965205 6.557133 4.761169 1.839126 2.147347 1.993236 Rpsa-ps10 -1.45276 -2.38842 2.892482 2.105718 2.4991 0.813954 1.27873 1.046342 Zfp58 -1.45768 -2.38261 3.100026 1.923934 2.51198 1.206223 0.902376 1.054299 RP23-421P10.1 -1.59163 -2.38031 2.369292 3.120174 2.744733 0.733752 1.572445 1.153098 Olfr1412 -4.01987 -2.37618 2.989666 10.89216 6.940911 5.439273 0.402802 2.921037 RP23-9O15.2 -4.45276 -2.37457 11.71768 3.66659 7.692133 3.140374 3.338377 3.239375 Cyp2e1 -1.20149 -2.36657 3.723605 0.437789 2.080697 1.181121 0.577283 0.879202 4930511A02Rik -2.04202 -2.36311 5.249583 1.830569 3.540076 1.270086 1.726033 1.498059 Gm9959 -1.07925 -2.35933 2.867451 0.878945 1.873198 0.461091 1.126813 0.793952 Krcc1 -3.26307 -2.3519 6.450944 4.902585 5.676764 1.508835 3.318554 2.413694 BC100451 -28.8889 -2.3452 63.758 36.97087 50.36444 23.4649 19.48621 21.47556 4933402N03Rik -1.80436 -2.33862 3.11321 3.191352 3.152281 1.444383 1.251466 1.347924 Spaca1 -17.2143 -2.33791 43.91008 16.25168 30.08088 11.30483 14.42829 12.86656

RP23-346B17.3 -1.11979 -2.33566 3.666576 0.249755 1.958166 0.524082 1.152674 0.838378 Sly -1.57789 -2.3266 3.188782 2.34586 2.767321 1.025524 1.353332 1.189428 Gm21103 -4.92144 -2.32408 13.71372 3.562903 8.638313 3.443051 3.990692 3.716872 RP23-464D14.3 -1.52254 -2.32349 2.528024 2.817828 2.672926 1.108666 1.192115 1.150391 RP24-188N22.2 -9.34998 -2.32137 19.62351 13.22842 16.42597 6.336139 7.815846 7.075992 1700024G13Rik -20.3331 -2.31776 53.1523 18.37394 35.76312 13.85591 17.00411 15.43001 Gm6650 -4.26861 -2.3106 12.44586 2.605343 7.5256 3.221632 3.292349 3.25699 Gm11802 -1.96697 -2.30937 4.138702 2.799704 3.469203 1.927687 1.076771 1.502229 4930503B20Rik -25.1812 -2.30441 62.87756 26.09415 44.48586 17.95264 20.65667 19.30466 RP23-313G19.1 -2.06965 -2.29984 5.605543 1.718239 3.661891 1.673995 1.510486 1.59224 4930415O20Rik -10.7379 -2.28363 30.13103 8.075118 19.10307 7.413307 9.317109 8.365208 Gm16541 -1.59705 -2.2836 3.666991 2.015492 2.841242 1.05732 1.431068 1.244194 Gm15343 -1.19695 -2.28231 2.475416 1.785358 2.130387 1.592206 0.274661 0.933433 1700047I17Rik2 -5.31499 -2.28231 8.870096 10.04963 9.459863 4.477586 3.812157 4.144872 Rpl36-ps3 -2.76014 -2.27529 7.766012 2.082918 4.924465 2.497578 1.831071 2.164325 RP23-168P24.1 -2.52172 -2.27355 6.63191 2.371654 4.501782 2.26615 1.693978 1.980064 Gm6370 -5.78722 -2.27094 15.17841 5.50303 10.34072 3.411753 5.695239 4.553496 Hist2h2aa1 -5.17046 -2.26998 9.178014 9.305502 9.241758 4.313999 3.828602 4.071301 Gm5152 -5.36097 -2.26643 11.73048 7.457719 9.5941 4.471189 3.995063 4.233126 4930442L01Rik -4.30779 -2.26512 11.38099 4.044705 7.712846 3.076663 3.73344 3.405051 Dnajc5b -44.7231 -2.26365 113.3998 46.83055 80.11518 33.99405 36.79012 35.39208 Iqcf1 -19.5815 -2.26286 44.90301 25.27142 35.08721 14.48832 16.52303 15.50567 Arl13a -3.5854 -2.26224 7.926688 4.925126 6.425907 2.549252 3.131762 2.840507 Gm6664 -3.33602 -2.25848 10.75294 1.220758 5.986849 1.921214 3.380438 2.650826 Gm9059 -2.86822 -2.25699 9.675687 0.624388 5.150037 3.603064 0.960562 2.281813 RP24-317K11.3 -4.09802 -2.25028 10.77291 3.978514 7.375711 3.005443 3.549931 3.277687 Speer4a -1.84095 -2.24625 3.787522 2.848768 3.318145 1.453509 1.500878 1.477193 Srp54b -14.5291 -2.24549 30.67041 21.71872 26.19457 10.92284 12.40801 11.66543 Gm3005 -1.0378 -2.23581 2.548921 1.20623 1.877576 0.920413 0.759139 0.839776 Gm17019 -3.67698 -2.23058 8.019978 5.309981 6.664979 2.269744 3.706264 2.988004 1700003G13Rik -1.75907 -2.22603 3.960006 2.427682 3.193844 2.122588 0.746952 1.43477 Gm9 -1.41688 -2.22245 3.193856 1.957994 2.575925 1.027156 1.290938 1.159047 Gm2174 -1.2961 -2.21878 3.013676 1.705417 2.359546 0.596437 1.530447 1.063442 1700016D06Rik -4.85277 -2.21641 11.44668 6.237678 8.842178 4.053142 3.92567 3.989406 4930527A07Rik -1.00025 -2.21153 2.834108 0.817624 1.825866 1.179536 0.471689 0.825613 4930570N19Rik -1.22777 -2.20785 2.469757 2.018779 2.244268 0.635426 1.397566 1.016496 Adam20 -8.44284 -2.20077 20.84135 10.10668 15.47402 6.390096 7.67225 7.031173 Elfn1 -6.14925 -2.19864 15.29219 7.266655 11.27942 5.17687 5.083486 5.130178 Gm10263 -2.28802 -2.19725 4.970248 3.427887 4.199068 0.599419 3.222685 1.911052 Spata9 -17.9808 -2.19709 41.15323 24.8492 33.00121 14.51584 15.52498 15.02041 Zfp944 -1.75538 -2.1956 3.615213 2.831947 3.22358 1.421036 1.515369 1.468202 Mrap -1.15534 -2.19079 1.909675 2.341453 2.125564 0.859822 1.080632 0.970227 Fam183b -23.1677 -2.18711 59.16962 26.19785 42.68373 17.02053 22.0116 19.51607

Izumo2 -20.811 -2.18131 46.81483 30.04095 38.42789 13.22661 22.00724 17.61692 Lyzl1 -14.956 -2.17941 39.87654 15.39713 27.63684 11.33653 14.02522 12.68087 Ckmt2 -3.06652 -2.17878 7.33747 3.998433 5.667952 2.541278 2.661583 2.60143 4930427A07Rik -3.48841 -2.17426 6.920622 5.997695 6.459159 2.717318 3.22417 2.970744 Gm7361 -1.84534 -2.17075 4.325111 2.517981 3.421546 1.420993 1.731416 1.576205 CH25-211A23.1 -1.15792 -2.1697 2.854534 1.441154 2.147844 1.188037 0.791814 0.989926 2310031A07Rik -1.57914 -2.16968 2.16507 3.693347 2.929208 1.634779 1.06535 1.350064 1700019B21Rik -1.42229 -2.16854 2.635253 2.643615 2.639434 1.078646 1.355651 1.217149 4932438H23Rik -1.88275 -2.1663 4.521364 2.472705 3.497035 1.694692 1.533881 1.614287 Gm20390 -3.03503 -2.16441 5.172117 6.110933 5.641525 1.754102 3.458894 2.606498 Gm5800 -5.20706 -2.164 10.62989 8.731053 9.680472 3.982849 4.963966 4.473408 Gm7853 -5.45029 -2.16348 8.027055 12.24246 10.13475 8.814983 0.553937 4.68446 Zfp37 -13.8425 -2.16059 34.17718 17.36227 25.76973 11.33246 12.52191 11.92718 1700003E24Rik -8.98912 -2.15959 23.58068 9.901466 16.74107 5.298156 10.20576 7.751958 RP23-380A10.3 -1.48724 -2.15678 4.269137 1.276681 2.772909 0.80369 1.767648 1.285669 Tnp1 -657.917 -2.15031 1663.81 795.9183 1229.864 582.9588 560.9362 571.9475 Tmco2 -61.2626 -2.15009 159.2951 69.76553 114.5303 49.93767 56.59766 53.26767 Cox11 -1.39073 -2.14524 2.881556 2.328619 2.605087 0.884596 1.544121 1.214359 Pdcd5 -10.3783 -2.14488 27.02797 11.85834 19.44316 9.644712 8.485091 9.064901 Pam16 -9.09369 -2.14025 21.20475 12.93291 17.06883 10.00197 5.948309 7.97514 Iqcf4 -66.1647 -2.13273 150.7593 98.39306 124.5762 56.13605 60.68692 58.41148 Rfpl3s -5.75315 -2.13203 13.84638 7.824205 10.83529 4.259436 5.904848 5.082142 RP24-285E3.4 -2.86608 -2.12763 7.305317 3.510195 5.407756 2.476413 2.606948 2.54168 Dpm1 -1.21389 -2.12327 2.376779 2.212328 2.294554 1.128563 1.032774 1.080668 1700013D24Rik -14.2274 -2.11964 33.79304 20.0761 26.93457 9.97164 15.4426 12.70712 Msr1 -1.21838 -2.11945 2.727234 1.886281 2.306758 1.319381 0.85737 1.088375 Nqo2 -1.41814 -2.11548 3.423768 1.955178 2.689473 0.950261 1.592396 1.271329 H2-Eb1 -2.16205 -2.11519 5.981928 2.219634 4.100781 1.873184 2.00428 1.938732 Taf13 -1.51951 -2.11512 3.337785 2.426502 2.882144 1.025947 1.699327 1.362637 4921511C20Rik -1.48213 -2.11148 2.785319 2.845901 2.81561 0.970417 1.696534 1.333475 Tctex1d2 -5.20514 -2.11144 14.02766 5.749077 9.888371 4.096657 5.269799 4.683228 Cst12 -4.35034 -2.10828 11.6668 4.884522 8.275662 3.019973 4.830672 3.925322 Prm2 -1897.53 -2.10581 4195.836 3031.162 3613.499 1758.161 1673.771 1715.966 Ccdc147 -18.1775 -2.10365 49.31244 19.9832 34.64782 16.36082 16.57981 16.47032 RP23-76E22.1 -2.02692 -2.10262 4.922927 2.807443 3.865185 1.840967 1.83557 1.838268 RP24-421H8.2 -1.70312 -2.10166 4.028479 2.46966 3.249069 2.332033 0.759868 1.54595 4930403D09Rik -26.7377 -2.10143 62.07918 39.94703 51.01311 23.22158 25.32933 24.27546 Rps27a -1.87343 -2.09944 4.48948 2.665362 3.577421 1.580618 1.827357 1.703987 Samd10 -1.76225 -2.09944 3.883006 2.847238 3.365122 2.056829 1.148907 1.602868 2210011C24Rik -1.24528 -2.09542 2.176462 2.587695 2.382079 1.527182 0.746424 1.136803 Ttc23l -15.4068 -2.09439 41.71255 17.2571 29.48482 15.44498 12.71107 14.07802 Dnajb3 -19.5415 -2.09379 38.3941 36.42062 37.40736 15.91226 19.81947 17.86586 4930486I03Rik -1.30241 -2.08845 3.547924 1.450037 2.498981 0.608547 1.784597 1.196572

Adam25 -7.98255 -2.08778 19.17197 11.4699 15.32094 6.726303 7.950481 7.338392 Cpxcr1 -3.56395 -2.08631 7.532448 6.157024 6.844736 2.816818 3.744746 3.280782 4933403O08Rik -1.42131 -2.08584 3.086233 2.374294 2.730264 1.362316 1.255591 1.308953 Fabp12 -3.24021 -2.08582 8.225787 4.222849 6.224318 2.688459 3.279765 2.984112 RP24-479J14.1 -2.98897 -2.08299 5.228986 6.268788 5.748887 1.793772 3.726069 2.759921 Gm595 -1.8497 -2.08129 5.182221 1.938485 3.560353 1.723117 1.698184 1.710651 Upf3b -1.19558 -2.08096 3.146021 1.457216 2.301619 0.95931 1.252766 1.106038 Oxct2b -2.30996 -2.07973 3.16907 5.72961 4.44934 1.796529 2.482235 2.139382 Gm21655 -3.08147 -2.07808 7.709326 4.170179 5.939753 3.791944 1.924629 2.858287 1700001L19Rik -16.6251 -2.0753 39.01906 25.15301 32.08603 16.04639 14.87542 15.46091 4930468A15Rik -1.31402 -2.0727 2.229922 2.848029 2.538975 1.135489 1.314428 1.224958 Spata19 -58.8978 -2.07003 146.617 81.26457 113.9408 56.66919 53.41675 55.04297 Zc3h10 -3.60058 -2.06915 8.332874 5.603678 6.968276 3.828405 2.906992 3.367698 Dsg1a -3.03116 -2.0685 7.9982 3.73776 5.86798 2.783091 2.890554 2.836823 1700015F17Rik -8.08808 -2.06626 20.92239 10.4247 15.67354 6.916371 8.254559 7.585465 Cox7b2 -8.38682 -2.06578 15.64665 16.86537 16.25601 5.197912 10.54048 7.869197 Golga7b -1.39969 -2.06322 3.711468 1.720827 2.716147 1.103348 1.529569 1.316459 Gm16277 -1.06141 -2.05622 3.09702 1.035616 2.066318 0.81492 1.194898 1.004909 Cst8 -43.7857 -2.05582 103.1341 67.37895 85.25652 38.01707 44.92456 41.47081 Gm15130 -3.31273 -2.05409 4.638523 8.272428 6.455476 2.441072 3.84442 3.142746 Fam24a -3.59889 -2.05254 6.028602 8.007644 7.018123 2.100389 4.738081 3.419235 Gm15341 -2.34749 -2.05236 6.454186 2.702164 4.578175 2.53133 1.930048 2.230689 RP23-80J7.2 -2.30382 -2.05124 3.727686 5.263019 4.495352 2.252362 2.130701 2.191531 1700017N19Rik -11.3051 -2.04865 32.33279 11.83893 22.08586 9.431956 12.12947 10.78071 Sox5os3 -1.29839 -2.04801 3.231663 1.84295 2.537307 0.918464 1.559363 1.238914 Timm8b -13.364 -2.04299 30.23882 22.1154 26.17711 12.28411 13.34215 12.81313 4921513D11Rik -2.11073 -2.04182 4.708073 3.56541 4.136742 2.093067 1.958946 2.026007 Gm13857 -1.67945 -2.03487 3.759066 2.845551 3.302309 1.303541 1.942181 1.622861 Epyc -2.60441 -2.03429 6.586403 3.658555 5.122479 2.15332 2.88281 2.518065 4930525G20Rik -1.72294 -2.03292 4.180699 2.601231 3.390965 1.364596 1.971451 1.668023 Atp6v1b1 -3.15857 -2.03152 7.161183 5.280062 6.220622 3.112247 3.011855 3.062051 1700092E19Rik -1.44997 -2.023 3.801869 1.932806 2.867337 1.610379 1.224358 1.417368 Lgals1 -5.99838 -2.02213 8.736764 14.99701 11.86689 5.694479 6.042533 5.868506 RP24-80P7.1 -2.69109 -2.02087 6.441853 4.212458 5.327156 2.301916 2.970216 2.636066 Galntl5 -57.7131 -2.01285 140.6188 88.76971 114.6942 50.32613 63.63618 56.98115 Klk1b7-ps -1.8038 -2.00969 4.862199 2.318378 3.590288 2.171807 1.401167 1.786487 Plcz1 -31.3318 -2.00899 78.9178 45.85101 62.38441 27.25718 34.84804 31.05261 Hmga1-rs1 -16.4658 -2.0081 33.41391 32.18476 32.79933 14.45093 18.21611 16.33352 Eif4a-ps4_1 -1.17259 -2.0078 2.917415 1.754776 2.336096 1.132983 1.194036 1.163509 Tmem156 -1.69633 -2.00657 3.009546 3.753626 3.381586 1.902387 1.468119 1.685253 Tmco5 -80.0041 -2.00604 209.91 109.1453 159.5276 70.58303 88.46398 79.5235 4930570D08Rik -15.792 -2.00541 38.51308 24.48491 31.499 14.37382 17.04017 15.70699 Spaca7 -20.9715 -2.00484 65.51616 18.16795 41.84206 20.95209 20.78903 20.87056

4930405D11Rik -1.45864 -2.00288 3.991098 1.835058 2.913078 1.026842 1.882043 1.454442 1700013H16Rik -4.24191 -2.00229 10.50696 6.441292 8.474125 3.868798 4.595629 4.232213 Bcmo1 -3.706 -2.00029 9.564746 5.257083 7.410914 3.677127 3.732706 3.704916 Spata33 -25.8417 -1.9999 76.46074 26.91134 51.68604 27.42515 24.26345 25.8443 1700008F21Rik -9.62924 -1.99691 26.4564 12.12032 19.28836 8.679965 10.63826 9.659114 Gm6960 -1.04002 -1.99595 2.916812 1.251706 2.084259 1.125669 0.962817 1.044243 Mbd3l1 -13.6135 -1.99557 37.65228 16.92282 27.28755 10.58216 16.76601 13.67408 Tgif2lx2 -4.09859 -1.99528 11.21925 5.213939 8.216594 5.761404 2.474612 4.118008 Gm10501 -3.67344 -1.99386 6.48109 8.258092 7.369591 5.394769 1.997532 3.696151 Gm14145 -8.41694 -1.99271 28.61806 5.173321 16.89569 7.787725 9.169772 8.478748 1700084M14Rik -6.90291 -1.99 21.00574 6.745396 13.87557 7.184447 6.760877 6.972662 Cytl1 -1.23811 -1.98951 3.922873 1.055817 2.489345 1.600091 0.902376 1.251234 4930571N24Rik -3.1403 -1.98823 8.442866 4.193134 6.318 3.230812 3.124579 3.177696 1700017D01Rik -15.6966 -1.98814 40.73247 22.43081 31.58164 14.8849 16.88518 15.88504 BC061237 -8.00665 -1.9874 23.84226 8.388601 16.11543 6.734294 9.483276 8.108785 Pnp2 -1.11693 -1.98723 2.581124 1.915486 2.248305 0.917478 1.345276 1.131377 Bik -1.31643 -1.98632 2.272979 3.029239 2.651109 1.271302 1.39806 1.334681 Ccdc169 -7.37187 -1.98609 20.45408 9.241348 14.84771 7.605374 7.346304 7.475839 Fmr1nb -1.15506 -1.98496 3.679946 0.975564 2.327755 1.407387 0.938008 1.172698 Gm27252 -2.64719 -1.9846 6.36878 4.302788 5.335784 1.086812 4.290375 2.688594 1700006E09Rik -15.4114 -1.98292 45.42825 16.75283 31.09054 13.29538 18.06289 15.67914 Gm20604 -2.86403 -1.98228 5.468162 6.091297 5.779729 3.881746 1.949648 2.915697 Gm13942 -13.5039 -1.98115 27.81427 26.71994 27.26711 13.46387 14.06262 13.76325 4933415F23Rik -1.34829 -1.97723 2.82559 2.630421 2.728005 1.27894 1.480484 1.379712 RP24-178B2.1 -1.9306 -1.97673 5.009001 2.805393 3.907197 2.028557 1.924629 1.976593 Zfp72 -1.25737 -1.97365 3.203659 1.89386 2.54876 1.287886 1.294901 1.291393 Bbs5 -8.06611 -1.97364 19.89624 12.80492 16.35058 7.86967 8.699271 8.284471 Gm362 -7.13215 -1.97337 15.94022 12.97864 14.45943 6.942044 7.712519 7.327282 Tmem243 -3.1464 -1.97315 8.017543 4.74168 6.379611 2.730146 3.736274 3.23321 RP24-454N4.1 -10.8766 -1.96838 32.39187 11.82473 22.1083 11.19275 11.27066 11.23171 Proc -2.8965 -1.9682 7.617238 4.158995 5.888116 3.176996 2.806239 2.991618 4930544L04Rik -1.27231 -1.96817 2.996813 2.176097 2.586455 0.954429 1.67386 1.314145 Gm10704 -1.5163 -1.96781 2.610153 3.5559 3.083027 1.492329 1.641126 1.566727 Kcnmb3 -1.70755 -1.96764 2.976971 3.967462 3.472217 1.332042 2.197285 1.764663 Ccdc150 -14.2523 -1.96275 38.79139 19.3207 29.05604 14.32588 15.28165 14.80377 4930441J16Rik -1.10293 -1.96243 3.612099 0.885759 2.248929 0.929332 1.362657 1.145994 4922502D21Rik -156.015 -1.96111 418.5689 218.1186 318.3437 144.1487 180.5081 162.3284 Cfhr3 -2.87869 -1.9591 8.679661 3.080618 5.880139 1.909913 4.092982 3.001447 Tcte3 -47.178 -1.95863 122.1669 70.6168 96.39183 41.98975 56.43788 49.21381 Gm15756 -23.009 -1.95813 59.0308 35.01605 47.02343 23.02753 25.00136 24.01445 Dynlt1b -196.456 -1.95669 503.82 299.7914 401.8057 186.8501 223.8484 205.3492 1700061I17Rik -1.18732 -1.95586 3.174313 1.684608 2.429461 1.188477 1.295806 1.242141 Otud6b -3.99929 -1.95471 8.887234 7.489332 8.188283 3.681108 4.696883 4.188995

4930473A02Rik -1.37532 -1.9528 3.042469 2.595041 2.818755 1.701686 1.185193 1.443439 Gm7347 -1.90748 -1.95257 4.729618 3.090249 3.909934 1.921342 2.083569 2.002456 Gm3404 -3.4292 -1.95221 9.81404 4.24694 7.03049 3.235801 3.96678 3.60129 Ccdc175 -17.6699 -1.9521 43.08808 29.36948 36.22878 17.36719 19.7505 18.55885 Pdzk1ip1 -14.803 -1.95079 32.33946 28.40507 30.37226 18.13765 13.00081 15.56923 Tmem95 -1.98289 -1.94964 4.914087 3.227766 4.070926 1.693274 2.482808 2.088041 4930589P08Rik -8.49252 -1.94913 18.22577 16.65456 17.44016 8.571026 9.324272 8.947649 Lrrc18 -30.7259 -1.94739 75.45117 50.86449 63.15783 29.55043 35.31351 32.43197 Sult1e1 -1.16011 -1.94528 2.144899 2.629861 2.38738 1.617479 0.837061 1.22727 1700019D03Rik -24.7805 -1.94487 67.94633 34.06776 51.00705 26.2886 26.16444 26.22652 Gm6408 -1.56408 -1.94471 4.616571 1.822834 3.219703 1.761516 1.549722 1.655619 Aif1 -46.7821 -1.9442 125.9797 66.67823 96.32898 47.46051 51.63333 49.54692 Nmnat1 -2.56502 -1.94256 6.321232 4.251468 5.28635 2.230304 3.212363 2.721333 Chrm4 -1.59793 -1.94177 3.494705 3.094621 3.294663 1.623426 1.770035 1.696731 Arl4aos -2.68441 -1.93849 6.35401 4.735495 5.544753 1.843133 3.877562 2.860347 Slxl1 -4.82625 -1.9366 10.72348 9.234948 9.979214 3.202375 7.103551 5.152963 Speer4e -2.8987 -1.93561 8.429732 3.564033 5.996883 2.467975 3.728394 3.098184 Ldhal6b -2.03928 -1.93534 4.643484 3.795585 4.219534 1.866702 2.493804 2.180253 Gm13248 -2.52452 -1.93347 6.066626 4.391281 5.228953 2.444689 2.96418 2.704434 RP23-84H3.1 -1.38025 -1.93238 4.007262 1.713944 2.860603 1.378663 1.582045 1.480354 Rpl9-ps6 -1.0967 -1.93203 2.562447 1.984305 2.273376 0.954212 1.399139 1.176676 Gm12551 -1.49944 -1.92986 4.492734 1.731256 3.111995 1.651292 1.573813 1.612552 Sycp1 -8.60927 -1.928 19.54947 16.22354 17.88651 7.098151 11.45631 9.277232 Gm6410 -1.30467 -1.92755 3.457808 1.964702 2.711255 1.301905 1.511255 1.40658 Psmd10 -2.9457 -1.92606 7.041748 5.211447 6.126597 2.652296 3.709508 3.180902 Gm5458 -3.18047 -1.92548 9.647645 3.586446 6.617046 2.178505 4.69464 3.436572 Rmdn2 -11.3434 -1.92062 27.92661 19.40314 23.66488 11.37632 13.26663 12.32147 Gm7536 -1.91129 -1.92038 4.595273 3.38056 3.987917 2.246343 1.906914 2.076628 Gm20825 -1.32438 -1.91937 3.572619 1.957195 2.764907 1.662336 1.218722 1.440529 RP24-539M22.1 -12.4235 -1.91649 31.71574 20.24238 25.97906 9.082103 18.029 13.55555 4930443O20Rik -1.77807 -1.91604 3.874933 3.563293 3.719113 1.993909 1.888173 1.941041 Stra8 -2.13519 -1.91588 4.496792 4.436164 4.466478 2.127721 2.53486 2.33129 Smok3c -3.06909 -1.91524 9.559194 3.285672 6.422433 3.009957 3.69672 3.353338 1700093K21Rik -24.5732 -1.91297 70.81117 32.16669 51.48893 24.49046 29.34092 26.91569 0610009B22Rik -3.86296 -1.91284 8.404101 7.785449 8.094775 4.324464 4.139174 4.231819 4930406D18Rik -8.5797 -1.91146 22.87133 13.11442 17.99288 8.416748 10.4096 9.413176 Rp2h -1.70515 -1.90999 4.713083 2.44484 3.578961 1.678979 2.068636 1.873808 Gm10354 -2.24618 -1.90891 6.519615 2.91535 4.717483 2.1214 2.821205 2.471303 Klhl3 -1.25172 -1.90882 2.855392 2.402645 2.629018 1.56652 1.188078 1.377299 Nrm -9.78975 -1.90773 25.87258 15.27662 20.5746 10.958 10.61172 10.78486 Tmem225 -8.53982 -1.90698 23.36842 12.54245 17.95544 9.537581 9.293652 9.415616 Nmb -1.84102 -1.90442 4.58322 3.169967 3.876594 1.965308 2.105847 2.035577 Slit1 -1.0484 -1.9043 2.833066 1.582428 2.207747 1.23508 1.083613 1.159347

Snrpd1 -6.01561 -1.90366 14.69697 10.64816 12.67256 6.803439 6.510474 6.656957 Gm3402 -1.8543 -1.89978 5.034846 2.795423 3.915134 1.344262 2.777404 2.060833 Gm26844 -1.79341 -1.89908 5.538909 2.037377 3.788143 1.246934 2.742525 1.994729 1700112K13Rik -1.15509 -1.89871 2.924718 1.955999 2.440358 0.9406 1.62994 1.28527 1700125H03Rik -2.02837 -1.89859 5.223509 3.347831 4.28567 1.603541 2.911053 2.257297 Adam26a -8.19294 -1.8972 19.13266 15.51662 17.32464 8.693861 9.569533 9.131697 Zfp458 -1.01783 -1.89617 2.538204 1.768979 2.153591 0.910814 1.360704 1.135759 Ugt1a9 -1.07042 -1.89236 2.765977 1.773945 2.269961 0.793751 1.605322 1.199537 Lrp2bp -8.4827 -1.89077 21.99833 14.01288 18.0056 9.340874 9.704928 9.522901 Spanxn4 -29.5133 -1.88928 88.12496 37.27726 62.70111 31.88398 34.49166 33.18782 Rom1 -2.18345 -1.88854 5.313052 3.968502 4.640777 2.553226 2.361433 2.457329 Speer4f -7.8006 -1.88796 23.89162 9.279313 16.58547 8.542673 9.027062 8.784867 Hist1h4m -6.72296 -1.88417 9.041029 19.61229 14.32666 8.648331 6.559059 7.603695 1700024P04Rik -30.2894 -1.88342 85.44228 43.70972 64.576 34.76878 33.80438 34.28658 Zfp474 -18.8608 -1.88313 42.78147 37.65348 40.21748 20.80884 21.90454 21.35669 Gm11335 -1.23225 -1.88116 2.933827 2.327578 2.630702 1.332042 1.464857 1.398449 Gm16226 -5.09613 -1.88107 17.77075 3.989484 10.88012 4.722369 6.845615 5.783992 Apoh -6.36435 -1.87994 16.37001 10.82415 13.59708 5.753518 8.711945 7.232732 4921530L21Rik -11.9851 -1.87885 34.34243 16.90221 25.62232 13.741 13.53345 13.63723 4933434M16Rik -3.12659 -1.87873 8.218808 5.150509 6.684658 3.67111 3.445033 3.558071 4930513O06Rik -2.11888 -1.87729 4.520515 4.547778 4.534146 2.534856 2.295671 2.415263 1700029I15Rik -4.35349 -1.86968 10.00368 8.714968 9.359322 5.418475 4.593194 5.005835 Iqcf3 -36.9254 -1.86885 100.1293 58.71987 79.42459 34.9437 50.05474 42.49922 Spaca4 -1.1284 -1.8681 2.109016 2.747482 2.428249 1.356535 1.24316 1.299847 Sf3b6 -1.33755 -1.86804 3.092475 2.664427 2.878451 1.505267 1.576527 1.540897 Gm21987 -1.43614 -1.86721 3.678637 2.505766 3.092201 1.577418 1.734699 1.656058 Gm8897 -2.23602 -1.86564 3.815875 5.822314 4.819095 2.127146 3.039011 2.583078 Ercc5 -1.28627 -1.86444 3.439765 2.108748 2.774257 1.434016 1.541954 1.487985 Adam3 -128.595 -1.86301 324.5395 230.6641 277.6018 139.1574 158.8564 149.0069 1700120B22Rik -2.36847 -1.86221 7.02958 3.201332 5.115456 2.842072 2.651896 2.746984 Gm732 -1.29528 -1.86075 3.639457 1.960725 2.800091 1.2414 1.768229 1.504815 Lat2 -1.40911 -1.85956 3.591458 2.505433 3.048445 1.234683 2.043986 1.639335 1700034E13Rik -18.5971 -1.85951 54.58397 25.88405 40.23401 17.98256 25.2912 21.63688 Gm11608 -4.15967 -1.85898 12.04524 5.95929 9.002267 3.805834 5.879356 4.842595 Poglut1 -1.6035 -1.85865 3.581235 3.360674 3.470955 2.18766 1.547251 1.867455 Ptrh1 -1.84224 -1.85857 4.351704 3.624198 3.987951 2.587799 1.703622 2.145711 Gm5861 -2.86298 -1.85734 6.462229 5.942506 6.202367 3.390873 3.287908 3.33939 Gm16445 -1.24623 -1.85651 3.001988 2.400481 2.701235 1.679044 1.230972 1.455008 Stat4 -37.5903 -1.85346 95.18981 68.07969 81.63475 40.7611 47.32788 44.04449 Gm6685 -1.82635 -1.8517 3.903336 4.038087 3.970712 3.138318 1.150411 2.144365 Pdcl2 -63.4038 -1.85168 170.9174 104.7809 137.8491 69.84008 79.05069 74.44538 Adam39 -7.90865 -1.85162 20.76875 13.62167 17.19521 7.735229 10.8379 9.286564 Pla2g10 -4.00888 -1.85087 12.57846 4.862252 8.720356 4.105062 5.317897 4.71148

Vash2 -15.8799 -1.8505 37.31691 31.78517 34.55104 17.13239 20.20998 18.67119 Als2cr11_1 -11.0418 -1.84836 28.69155 19.423 24.05727 11.56086 14.47008 13.01547 Ptpmt1 -4.41085 -1.84824 10.59922 8.622446 9.610831 4.86499 5.534963 5.199976 Spta1 -1.14463 -1.84615 3.096468 1.89829 2.497379 1.336028 1.369477 1.352752 Gm26706 -1.32509 -1.84583 2.873216 2.910181 2.891699 1.365968 1.767253 1.566611 4930524N10Rik -1.78495 -1.84474 4.563272 3.232683 3.897977 2.217654 2.0084 2.113027 4930533K18Rik -1.28521 -1.84453 4.071142 1.542867 2.807005 1.856818 1.186778 1.521798 K230010J24Rik -5.23802 -1.84388 14.31257 8.577588 11.44508 9.20595 3.208157 6.207054 1700008I05Rik -3.53027 -1.84344 10.28472 5.146978 7.715848 3.273853 5.097305 4.185579 Amer2 -10.8399 -1.84233 29.76114 17.65652 23.70883 13.8056 11.93228 12.86894 Ppp1r42 -6.52345 -1.84219 18.78528 9.753236 14.26926 7.123756 8.367861 7.745809 4931406G06Rik -1.84186 -1.84193 4.317208 3.741837 4.029522 2.058707 2.316626 2.187667 Slc7a6os -2.81547 -1.83984 6.416508 5.919194 6.167851 3.419635 3.285121 3.352378 2810428I15Rik -1.11324 -1.83977 2.557413 2.320378 2.438895 1.348857 1.302452 1.325654 RP24-425N5.7 -9.41727 -1.8381 27.596 13.71155 20.65378 10.77165 11.70136 11.2365 Adtrp -2.16082 -1.83594 4.966569 4.524845 4.745707 2.795217 2.374564 2.584891 Hrasls -5.87901 -1.83542 15.08771 10.74475 12.91623 6.585216 7.489224 7.03722 4933421I07Rik -1.83842 -1.83404 5.012458 3.072888 4.042673 2.418038 1.990463 2.204251 Acrv1 -5.89035 -1.83401 18.44934 7.456697 12.95302 6.919532 7.205793 7.062662 Gm14966 -1.4019 -1.83375 3.60219 2.564504 3.083347 1.719032 1.643854 1.681443 1700019A02Rik -12.0072 -1.83289 33.58276 19.2644 26.42358 12.88326 15.9494 14.41633 Map2 -2.22516 -1.83101 6.249069 3.556606 4.902838 2.518077 2.837275 2.677676 Gm9758 -1.94766 -1.82956 5.298307 3.292623 4.295465 1.820665 2.874952 2.347808 Gm15415 -4.25703 -1.82881 11.82405 6.962598 9.393322 5.503846 4.768732 5.136289 Smok2b -1.0923 -1.82246 3.354271 1.486508 2.420389 1.169725 1.486455 1.32809 Gsdmd -1.21468 -1.82051 2.623653 2.766501 2.695077 1.822557 1.138233 1.480395 Lyzl6 -11.1303 -1.81863 34.72416 14.72875 24.72646 12.67653 14.5158 13.59617 Rgsl1 -3.05132 -1.8186 8.486136 5.071473 6.778805 3.572281 3.882679 3.72748 RP23-153H17.1 -1.00742 -1.81849 3.142666 1.333808 2.238237 1.321676 1.139966 1.230821 Btg4 -1.64474 -1.81704 4.265087 3.050497 3.657792 2.148946 1.877162 2.013054 Calml3 -1.04147 -1.81512 2.875494 1.762822 2.319158 1.261049 1.294333 1.277691 Cldnd2 -8.79621 -1.81494 25.63492 13.54484 19.58988 11.56933 10.01802 10.79367 Tmem190 -14.3635 -1.81414 43.73352 20.27874 32.00613 17.28699 17.99824 17.64262 Tmem141 -1.44829 -1.8134 4.319066 2.138611 3.228838 1.602724 1.958364 1.780544 Sun3 -12.3656 -1.81332 37.01859 18.12022 27.5694 12.27447 18.1331 15.20379 Gm14773 -2.1541 -1.81323 6.818938 2.7869 4.802919 1.594906 3.702742 2.648824 1700007K13Rik -4.80017 -1.81184 12.07152 9.354166 10.71284 6.087362 5.737987 5.912675 Tctex1d1 -10.4569 -1.80998 28.88883 17.84515 23.36699 11.95536 13.86485 12.91011 Vav3 -1.01225 -1.80965 2.793437 1.731545 2.262491 1.197839 1.302637 1.250238 Scp2d1 -58.0398 -1.80923 168.1137 91.41033 129.762 61.91366 81.53083 71.72224 Fam3b -3.76075 -1.80864 9.577327 7.245535 8.411431 3.407775 5.893592 4.650683 4930526F13Rik -2.72453 -1.80684 6.22725 5.975399 6.101325 2.821204 3.932387 3.376795 Pdilt -17.471 -1.80598 51.10537 27.19019 39.14778 20.87737 22.4762 21.67679

Gm13166 -3.88248 -1.80519 10.69202 6.716588 8.704302 4.414983 5.228667 4.821825 1700012B09Rik -9.96866 -1.80462 25.60224 19.11358 22.35791 12.22258 12.55591 12.38925 Glrx2 -2.17588 -1.80428 5.983049 3.779456 4.881252 2.402632 3.008113 2.705372 Gm5862 -2.08706 -1.80288 5.954468 3.418617 4.686542 1.945376 3.253586 2.599481 Enkur -13.601 -1.80201 38.20818 22.91114 30.55966 15.16787 18.74941 16.95864 Hnrnph2 -1.07239 -1.8018 2.858262 1.961483 2.409873 1.166184 1.508779 1.337482 Spsb2 -6.02011 -1.80053 14.95121 12.12924 13.54022 7.989841 7.050395 7.520118 Tsacc -19.1193 -1.80045 48.60741 37.40261 43.00501 24.85001 22.92132 23.88566 Med31 -1.28987 -1.79784 3.501439 2.311677 2.906558 1.455236 1.77815 1.616693 Gm14147 -7.16204 -1.79762 25.54876 6.733883 16.14132 8.943213 9.015343 8.979278 Cypt15 -2.16092 -1.79627 5.56639 4.183035 4.874713 2.887374 2.540214 2.713794 Cdc25c -2.46493 -1.79563 6.769754 4.356237 5.562995 3.026432 3.169705 3.098069 Habp4 -30.6316 -1.7951 71.07445 67.23967 69.15706 36.32487 40.72598 38.52543 Mipepos -1.76776 -1.7938 4.430446 3.559009 3.994728 1.572246 2.881685 2.226966 4933413G19Rik -3.06779 -1.7923 8.151206 5.728381 6.939794 3.900219 3.843784 3.872001 Chl1 -30.5906 -1.79196 78.06691 60.36726 69.21708 36.89624 40.35679 38.62651 Cox20 -2.48171 -1.79083 5.80472 5.434929 5.619825 3.190565 3.085655 3.13811 Akr1c13 -1.66487 -1.79044 3.461423 4.080818 3.77112 2.140782 2.071726 2.106254 Gtsf1l -86.204 -1.78937 239.4426 151.3775 195.4101 112.6645 105.7477 109.2061 1700019L03Rik -17.7281 -1.78908 46.41273 33.977 40.19486 24.52934 20.40424 22.46679 Usp16 -5.11025 -1.78831 13.87926 9.306372 11.59282 6.308671 6.656467 6.482569 Fam154aos -1.87559 -1.78716 5.78741 2.72921 4.25831 2.386221 2.379226 2.382724 Fer1l5 -3.80324 -1.7862 11.91335 5.368056 8.640701 5.124466 4.550465 4.837466 Lyzl4os -3.09148 -1.78483 8.5865 5.474513 7.030507 3.019186 4.858861 3.939023 4921511H03Rik -3.20146 -1.78339 8.88867 5.687603 7.288136 3.877016 4.296331 4.086673 Cep83 -24.5664 -1.78141 64.29197 47.71797 56.00497 29.46908 33.40815 31.43862 Txndc8 -21.6948 -1.78099 55.17956 43.76737 49.47347 23.04783 32.50954 27.77868 4930428N03Rik -6.29816 -1.77923 18.30906 10.45232 14.38069 7.851016 8.314051 8.082533 Rpl34 -3.85523 -1.77912 15.57155 2.035217 8.803382 6.914416 2.981896 4.948156 Lyzl4 -9.16112 -1.7791 20.70937 21.1302 20.91979 12.2492 11.26813 11.75866 Gm4907 -5.57196 -1.77876 14.35646 11.0972 12.72683 7.186602 7.123147 7.154875 Gm355 -3.31953 -1.77775 9.047167 6.128154 7.58766 3.703131 4.833132 4.268131 Rmnd1 -7.6075 -1.77683 19.19499 15.60598 17.40048 9.132645 10.45332 9.792983 Proca1 -22.4413 -1.77646 62.57442 40.11241 51.34342 29.76999 28.03432 28.90216 Pldi -5.12542 -1.77605 13.54728 9.912609 11.72994 5.481204 7.727848 6.604526 C12orf54 -18.8765 -1.77595 58.28704 28.11992 43.20348 21.69565 26.95829 24.32697 Fam103a1 -3.31757 -1.77531 8.564471 6.628693 7.596582 4.165978 4.392046 4.279012 Rpf2 -2.0508 -1.77422 5.182723 4.216563 4.699643 2.64816 2.649533 2.648846 4930507D10Rik -1.54768 -1.7727 4.125694 2.975597 3.550645 1.717085 2.288838 2.002962 Zfp558 -1.3061 -1.77217 3.506239 2.488895 2.997567 1.543058 1.839876 1.691467 Mas1 -4.48437 -1.77168 11.73794 8.853195 10.29557 6.154338 5.468052 5.811195 Uqcr11 -17.1282 -1.77154 47.10492 31.55154 39.32823 21.22747 23.17261 22.20004 Dgka -26.8661 -1.77098 67.63732 55.78862 61.71297 34.26165 35.432 34.84683

Zscan12 -11.5301 -1.77055 32.16551 20.8217 26.4936 12.80626 17.12068 14.96347 Pebp4 -45.5296 -1.76899 135.9163 73.55607 104.7362 56.96483 61.44843 59.20663 Gm20499 -2.88574 -1.76532 7.136336 6.176374 6.656355 3.780119 3.761118 3.770619 Fam170b -44.4482 -1.76515 123.3943 81.68382 102.5391 59.83573 56.34596 58.09084 Eqtn -11.3045 -1.76513 34.75698 17.40135 26.07916 14.25192 15.29734 14.77463 Tmed6 -4.75067 -1.7646 8.422929 13.505 10.96397 6.112266 6.31433 6.213298 4933411G06Rik -2.84314 -1.76431 8.206745 4.919343 6.563044 3.225835 4.213971 3.719903 Leng8 -8.52491 -1.76404 27.29709 12.06814 19.68261 10.59839 11.71702 11.15771 Defb33 -5.79176 -1.76396 17.58342 9.162592 13.37301 7.492735 7.669768 7.581252 Ccdc81 -23.1396 -1.7637 65.2679 41.60972 53.43881 30.29321 30.30521 30.29921 Gfy -1.70906 -1.76355 6.007592 1.887134 3.947363 1.948031 2.528575 2.238303 Hist1h1a -91.5645 -1.76291 184.4972 238.6707 211.5839 113.8149 126.2241 120.0195 Gm8094 -1.07037 -1.76274 3.243193 1.704205 2.473699 1.745467 1.061189 1.403328 2010109I03Rik -1.79508 -1.7627 5.390581 2.906749 4.148665 2.577755 2.12941 2.353583 1700016C15Rik -9.48993 -1.76259 23.78341 20.08528 21.93435 10.92691 13.96191 12.44441 C230052I12Rik -1.16219 -1.76229 2.554609 2.818986 2.686798 1.314515 1.734699 1.524607 Rbp4 -1.07867 -1.76202 2.874083 2.114348 2.494216 1.340258 1.490833 1.415546 1700121C08Rik -2.10006 -1.76195 4.81791 4.894571 4.85624 2.656213 2.856146 2.75618 Gm14266 -1.6597 -1.76075 4.652338 3.030374 3.841356 1.963775 2.399531 2.181653 Gm11213 -5.2976 -1.75877 12.20604 12.35275 12.27939 6.106346 7.857235 6.98179 Thg1l -2.34729 -1.75872 7.016457 3.865611 5.441034 2.92254 3.264954 3.093747 Gm5941 -1.52386 -1.75871 6.656582 0.408082 3.532332 1.819664 2.197285 2.008475 Papd5 -12.146 -1.75849 30.97596 25.3429 28.15943 15.31025 16.71663 16.01344 Gm15104 -29.0259 -1.75848 79.35721 55.2316 67.2944 35.09661 41.44048 38.26855 Zranb1 -5.18568 -1.75651 11.20502 12.87588 12.04045 5.860641 7.848891 6.854766 Cox6c -3.00227 -1.75617 7.602723 6.342601 6.972662 3.949073 3.991703 3.970388 Gm13923 -1.08529 -1.75585 2.265087 2.777224 2.521155 1.040658 1.831071 1.435864 H2afb1 -60.0132 -1.75584 178.475 100.349 139.412 73.98731 84.81033 79.39882 Hoxd8 -1.68515 -1.75319 4.347155 3.497844 3.9225 2.424763 2.049945 2.237354 Ms4a14 -17.6183 -1.75285 50.39024 31.65069 41.02046 21.20453 25.59986 23.40219 Gm28037 -5.96694 -1.75283 19.37054 8.415436 13.89299 9.810455 6.041634 7.926045 Cstl1 -13.6551 -1.75113 38.98065 24.68839 31.83452 18.0988 18.25999 18.17939 Stard6 -67.7601 -1.75095 195.1317 120.8539 157.9928 88.01115 92.45419 90.23267 1700012B07Rik -27.0878 -1.75082 77.78141 48.54993 63.16567 36.3451 35.8106 36.07785 Shcbp1 -4.91826 -1.75057 14.20612 8.735861 11.47099 5.932335 7.173125 6.55273 BC061195 -9.12435 -1.74845 33.91742 8.71329 21.31535 11.73905 12.64296 12.191 Sfi1 -4.08501 -1.74813 7.693068 11.39757 9.545319 5.45575 5.46487 5.46031 Amph -1.52773 -1.74716 3.852604 3.292261 3.572432 1.914836 2.174576 2.044706 Yjefn3 -1.20445 -1.74705 3.229431 2.404046 2.816739 3.115791 0.108776 1.612284 Cmtm2a -109.366 -1.74691 280.0937 231.4855 255.7896 137.9606 154.8872 146.4239 Gm16001 -1.94616 -1.74603 2.957058 6.152618 4.554838 2.766548 2.450818 2.608683 Aven -10.6958 -1.74529 28.54836 21.54568 25.04702 13.93969 14.7628 14.35125 Gm11520 -1.71364 -1.74435 4.585645 3.446024 4.015835 2.093209 2.511183 2.302196

Ube2n -22.6058 -1.74413 59.38549 46.58343 52.98446 28.87359 31.88374 30.37866 Catsper4 -13.7352 -1.74351 37.1704 27.24689 32.20865 16.49044 20.4564 18.47342 Gpr113 -1.31417 -1.7425 4.534898 1.63332 3.084109 1.695436 1.844436 1.769936 Taf15 -3.75274 -1.74187 12.2596 5.362802 8.811203 4.907883 5.209048 5.058465 1700030M09Rik -1.32123 -1.74144 4.651151 1.555303 3.103227 1.251056 2.312931 1.781994 Mthfd2l -1.36795 -1.74112 3.002163 3.425331 3.213747 1.528719 2.162876 1.845797 Sel1l2 -5.22579 -1.74039 14.22209 10.34582 12.28396 6.06203 8.054305 7.058168 AI987944 -5.15522 -1.74018 13.50257 10.73755 12.12006 5.834852 8.094827 6.96484 Gm5616 -2.36326 -1.73963 6.983391 4.133542 5.558466 1.393997 4.99641 3.195204 1700101I11Rik -2.14652 -1.73913 5.060207 5.041011 5.050609 3.32257 2.485616 2.904093 Ergic2 -7.41872 -1.73885 20.07365 14.84552 17.45958 9.302709 10.77901 10.04086 RP24-531B21.1 -3.64068 -1.73859 10.25791 6.881879 8.569894 5.477555 4.38088 4.929217 Msantd2 -1.01564 -1.73839 3.134904 1.647303 2.391103 1.099851 1.651083 1.375467 Prame -1.25635 -1.73825 3.246111 2.670165 2.958138 1.427188 1.976394 1.701791 Adam26b -1.27597 -1.73814 3.681187 2.328018 3.004602 1.520355 1.9369 1.728628 Gm136 -16.74 -1.73711 44.99667 33.9041 39.45038 23.36407 22.05665 22.71036 Sycp3 -9.92889 -1.73699 30.89202 15.91003 23.40102 10.8123 16.13197 13.47213 Gm6760 -9.08668 -1.73673 32.24695 10.59391 21.42043 9.824911 14.84259 12.33375 Chchd3 -8.41482 -1.73631 25.05441 14.63195 19.84318 11.38999 11.46673 11.42836 4930557A04Rik -4.14973 -1.73587 10.27978 9.298067 9.788926 4.444154 6.834231 5.639193 Dynlt3 -1.179 -1.73584 3.255937 2.30655 2.781243 1.396162 1.808325 1.602243 4930471C04Rik -1.17005 -1.73528 4.201727 1.320959 2.761343 1.455236 1.727345 1.591291 Crisp2 -147.696 -1.73517 419.6455 277.5493 348.5974 189.5149 212.2878 200.9014 Prss40 -6.72353 -1.73474 18.07874 13.67016 15.87445 8.457005 9.844842 9.150924 RP24-247J24.1 -3.26535 -1.734 11.14077 4.287352 7.714062 3.45215 5.445278 4.448714 Dpy30 -11.7167 -1.73339 32.62682 22.75873 27.69278 15.07778 16.87443 15.9761 4930451I11Rik -8.479 -1.73309 25.89761 14.19266 20.04513 11.10502 12.02724 11.56613 Itga1 -2.59308 -1.7322 7.132688 5.136485 6.134586 3.205448 3.877562 3.541505 Gng10 -1.33117 -1.73204 2.907822 3.391365 3.149593 2.098422 1.538434 1.818428 H1fnt -114.05 -1.73172 310.3594 229.4737 269.9165 160.3423 151.3906 155.8664 Adam29 -2.41496 -1.73164 7.281705 4.14968 5.715693 2.506741 4.09472 3.30073 Tra2a -5.93186 -1.73161 17.07843 11.00124 14.03984 6.984475 9.231481 8.107978 Ufd1l -6.69597 -1.73129 19.45085 12.25375 15.8523 8.501904 9.810755 9.15633 1700015G11Rik -13.0762 -1.73095 41.78388 20.14711 30.9655 15.85365 19.925 17.88932 E330034G19Rik -1.85244 -1.73092 6.07997 2.693698 4.386834 1.840321 3.228467 2.534394 Pnpla8 -5.58928 -1.73083 15.29985 11.17443 13.23714 6.499905 8.79582 7.647863 Rbm4b -4.84864 -1.73031 12.68646 10.28915 11.48781 7.948189 5.33014 6.639164 Atp5j -4.05577 -1.72955 11.01632 8.213788 9.615056 4.940896 6.17767 5.559283 Nup62 -7.8216 -1.7295 18.37039 18.71651 18.54345 9.94476 11.49894 10.72185 Rps24 -50.9808 -1.7269 154.7655 87.46554 121.1155 71.00259 69.26679 70.13469 Ahsa2 -1.05029 -1.72684 3.351792 1.638811 2.495301 1.4549 1.43512 1.44501 Lemd1 -1.14732 -1.72659 3.140181 2.312566 2.726374 1.587437 1.570672 1.579055 Spata7 -24.4473 -1.72582 64.76473 51.49462 58.12967 30.21629 37.14841 33.68235

RP24-284M17.1 -2.27959 -1.72568 5.648009 5.193769 5.420889 3.027368 3.255237 3.141302 1700030C12Rik -15.6166 -1.72508 43.12827 31.18038 37.15433 17.58774 25.48775 21.53775 Lrrc69 -2.42491 -1.72497 7.204547 4.334957 5.769752 2.788992 3.900692 3.344842 Fam71d -28.2635 -1.72455 85.4086 49.13563 67.27212 36.60108 41.41609 39.00859 Gm7008 -1.32158 -1.72374 2.996335 3.298929 3.147632 1.691274 1.960832 1.826053 Fabp9 -276.003 -1.72284 831.3762 484.2944 657.8353 379.0836 384.5814 381.8325 Gm614 -6.67435 -1.72251 19.86654 11.95775 15.91214 8.596317 9.879265 9.237791 Clk4 -2.60426 -1.72225 7.748597 4.671435 6.210016 3.133067 4.078452 3.60576 Nphp1 -110.372 -1.72202 318.8768 207.5973 263.2371 150.2974 155.4328 152.8651 Mtap7d3 -1.91578 -1.72122 5.705642 3.438559 4.5721 2.363672 2.948968 2.65632 Spata1 -7.63205 -1.72095 23.35467 13.08163 18.21815 9.208379 11.96383 10.5861 Rpl23 -31.8873 -1.72065 80.87183 71.39818 76.13501 39.38554 49.10994 44.24774 Pinlyp -1.34539 -1.72016 3.702999 2.724149 3.213574 2.22301 1.513362 1.868186 4933402D24Rik -1.17023 -1.72012 3.445671 2.144867 2.795269 1.500252 1.749829 1.62504 Cetn3 -16.4959 -1.72011 45.52329 33.28379 39.40354 22.50071 23.31453 22.90762 Atp6v1e2 -45.0232 -1.71972 117.9507 97.20875 107.5797 67.21841 57.89463 62.55652 Atxn3 -4.62529 -1.7187 13.28854 8.833345 11.06094 6.385523 6.48579 6.435657 Ccdc70 -26.6954 -1.71837 86.00423 41.70853 63.85638 38.06175 36.26013 37.16094 BC055111 -8.93436 -1.71803 23.56263 19.1918 21.37721 11.75191 13.1338 12.44285 Sema4a -2.20277 -1.71798 6.567926 3.973627 5.270776 2.949416 3.186591 3.068004 Gm12695 -2.09691 -1.71759 5.237164 4.800963 5.019063 2.613013 3.231301 2.922157 Mrpl47 -2.86261 -1.71666 7.466271 6.247709 6.85699 3.645588 4.343171 3.99438 Cttnbp2nl -5.13534 -1.7159 14.12793 10.48934 12.30863 7.157061 7.189516 7.173288 4930444G20Rik -3.41616 -1.71573 11.93154 4.44678 8.189159 4.192213 5.353791 4.773002 RP23-421C24.1 -2.23183 -1.71557 7.770163 2.931384 5.350774 3.043548 3.194343 3.118945 4930428D18Rik -1.19901 -1.71555 4.247144 1.502142 2.874643 1.733639 1.617633 1.675636 1700092M07Rik -15.0873 -1.71475 49.53898 22.85258 36.19578 22.55708 19.65992 21.1085 Gm6812 -5.47655 -1.71388 18.46092 7.835171 13.14805 7.307201 8.035785 7.671493 Plac8 -1.80527 -1.71359 4.507409 4.162845 4.335127 2.409724 2.649992 2.529858 Bcas1 -1.34726 -1.71351 3.777059 2.693881 3.23547 1.587864 2.188554 1.888209 Cast -19.465 -1.71269 57.19048 36.36356 46.77702 27.38188 27.24208 27.31198 Cylc2 -5.76554 -1.71227 21.86227 5.858119 13.86019 8.132974 8.056332 8.094653 4933406C10Rik -2.25802 -1.71186 7.83554 3.024476 5.430008 2.683268 3.660716 3.171992 RP23-168F21.1 -1.24457 -1.71121 4.180123 1.808912 2.994517 0.948948 2.550938 1.749943 Akap14 -2.01475 -1.71094 5.616491 4.080818 4.848655 2.675977 2.991839 2.833908 Gm3409 -1.08784 -1.71082 3.553253 1.683249 2.618251 1.766052 1.294761 1.530406 Kcne3 -13.3551 -1.71056 34.77076 29.53011 32.15044 18.45708 19.13352 18.7953 Fam217a -36.0914 -1.71031 103.8149 69.98902 86.90195 49.17987 52.44125 50.81056 Olfr128 -1.06536 -1.71017 4.032352 1.098682 2.565517 0.634001 2.366307 1.500154 BC048502 -5.95756 -1.709 17.26261 11.45812 14.36037 7.012699 9.792913 8.402806 Dram1 -1.95146 -1.70852 5.508895 3.902576 4.705736 2.795319 2.713232 2.754276 Ccdc67 -31.1332 -1.70811 89.0828 61.11737 75.10008 45.9657 41.96808 43.96689 Paip2 -10.1756 -1.70798 29.10375 19.99294 24.54834 13.77653 14.96895 14.37274

100

Prss47 -6.50474 -1.70766 14.37538 17.01788 15.69663 8.449255 9.934533 9.191894 Fam229b -22.7866 -1.70721 67.34119 42.67279 55.00699 28.84624 35.59456 32.2204 Dapl1 -1.09355 -1.7066 3.431228 1.851093 2.64116 1.412474 1.682751 1.547613 Snrnp48 -1.01963 -1.70627 3.211736 1.714896 2.463316 1.665978 1.221392 1.443685 Gzmn -7.43747 -1.70437 20.04658 15.94645 17.99651 9.497612 11.62048 10.55905 Tsga10 -8.19408 -1.70376 22.94373 16.73085 19.83729 10.62472 12.66171 11.64321 Gm19872 -2.254 -1.70081 6.782543 4.158039 5.470291 3.490066 2.942507 3.216286 4930412O13Rik -5.57223 -1.70021 17.54523 9.515015 13.53012 7.76944 8.146342 7.957891 Adam5 -122.457 -1.70013 343.9979 250.7284 297.3631 157.3163 192.4959 174.9061 Stpg1 -9.62274 -1.70005 28.92801 17.80915 23.36858 13.99429 13.4974 13.74585 Ccdc30 -15.5539 -1.69906 46.5185 29.08888 37.80369 22.56326 21.93627 22.24976 Hyal5 -6.40156 -1.69831 20.38261 10.75489 15.56875 8.674941 9.659446 9.167193 Nup62cl -1.42179 -1.69824 3.796099 3.119981 3.45804 2.066036 2.006473 2.036255 Cnbd1 -1.75173 -1.69802 4.831148 3.691439 4.261294 1.98155 3.037569 2.509559 Tmem63c -1.62665 -1.69621 4.740515 3.185613 3.963064 2.609356 2.063481 2.336418 Cops2 -7.80298 -1.69609 22.34616 15.67936 19.01276 9.86364 12.55591 11.20978 Atp5l -19.8791 -1.69549 63.83024 33.09376 48.462 28.27005 28.8958 28.58292 1700025D23Rik -2.85281 -1.69548 8.371871 5.537564 6.954718 4.463757 3.740059 4.101908 Zc3h8 -1.25521 -1.69506 3.729981 2.392204 3.061093 1.771681 1.840091 1.805886 4930564B18Rik -1.63916 -1.69497 4.182386 3.813147 3.997767 1.37956 3.337648 2.358604 Dynll2 -24.5673 -1.69477 77.592 42.26391 59.92796 33.58586 37.1354 35.36063 Cdkn2aip -14.5369 -1.69423 42.14184 28.811 35.47642 19.59177 22.28729 20.93953 Penk -26.0935 -1.69414 74.71341 52.65604 63.68473 36.83088 38.35163 37.59125 Speer3 -1.90654 -1.69405 5.472693 3.834326 4.65351 2.765775 2.728173 2.746974 Naca -7.09399 -1.69258 19.29027 15.38342 17.33685 9.902256 10.58345 10.24285 Kcnmb4os1 -29.1367 -1.69148 80.97764 61.56885 71.27325 49.39386 34.87923 42.13654 4930479D17Rik -2.83386 -1.69058 6.926443 6.94842 6.937432 4.050127 4.157025 4.103576 Gm17028 -6.0638 -1.6904 16.5469 13.14673 14.84681 9.451064 8.114973 8.783018 Zfand6 -15.682 -1.68894 45.92563 30.96333 38.44448 21.02278 24.5021 22.76244 4933427I04Rik -9.81527 -1.68889 31.37711 16.74949 24.0633 13.31737 15.17868 14.24802 4430402I18Rik -16.2743 -1.68772 42.7102 37.16707 39.93864 20.88012 26.4485 23.66431 1190005I06Rik -1.00832 -1.68754 2.83965 2.110118 2.474884 1.715788 1.217332 1.46656 Ccsap -1.46513 -1.68673 4.5131 2.684167 3.598633 1.884692 2.382311 2.133502 Ube2dnl1 -1.17425 -1.68559 4.38994 1.384073 2.887006 1.532834 1.892681 1.712758 Fam83e -1.75874 -1.68499 4.995468 3.657127 4.326297 2.573617 2.561498 2.567558 Gm4419 -1.59473 -1.68495 3.88076 3.965168 3.922964 2.11478 2.541683 2.328232 Prm1 -210.474 -1.68477 571.0751 464.6042 517.8397 266.3516 348.3798 307.3657 Cylc1 -4.41128 -1.68476 12.79393 8.912864 10.8534 5.891324 6.992919 6.442122 Ccer1 -35.3876 -1.68384 107.6599 66.61259 87.13623 55.83452 47.66273 51.74862 Ccdc50 -1.97824 -1.68354 5.92227 3.822431 4.87235 2.685208 3.103007 2.894107 4933406K04Rik -9.05017 -1.68293 25.68892 18.91543 22.30218 12.90883 13.59519 13.25201 Nkain1 -1.20234 -1.68277 3.202255 2.724361 2.963308 1.303084 2.218857 1.760971 Zfp607 -1.0832 -1.68222 3.346055 1.995859 2.670957 1.512362 1.663156 1.587759

101

Gm14474 -3.9285 -1.68214 11.29602 8.079196 9.687607 4.238315 7.279893 5.759104 Gm20521 -18.6822 -1.68196 52.32234 39.83141 46.07687 27.53956 27.24986 27.39471 Ppp2r2b -6.63483 -1.68083 18.1941 14.56601 16.38006 8.859666 10.63078 9.745222 Gpr39 -1.25084 -1.68075 3.523683 2.65287 3.088277 1.809191 1.865688 1.83744 Fkbpl -25.683 -1.68048 66.12736 60.72355 63.42546 35.35494 40.13005 37.7425 Gm26611 -3.53934 -1.68007 10.20959 7.277893 8.743739 4.810622 5.598178 5.2044 Taco1 -1.31215 -1.67938 3.099666 3.387404 3.243535 2.210438 1.652333 1.931386 Aldh3b1 -7.86573 -1.67783 22.91284 16.02728 19.47006 12.05231 11.15635 11.60433 Ube2dnl2 -1.08151 -1.67713 3.511952 1.845431 2.678691 1.774861 1.419511 1.597186 E2f5 -2.61258 -1.67688 7.397892 5.546743 6.472318 3.25213 4.467352 3.859741 Ubl5 -2.61511 -1.67644 7.954116 5.008063 6.48109 3.854394 3.877562 3.865978 Klk1b8 -3.47184 -1.6764 10.9574 6.251871 8.604635 4.535765 5.729823 5.132794 4930579D09Rik -1.34205 -1.67611 3.974079 2.67994 3.32701 1.533694 2.436222 1.984958 Cep290 -5.40439 -1.67567 15.07417 11.73172 13.40294 7.082816 8.914291 7.998553 1500002C15Rik -1.7989 -1.67524 4.738584 4.187372 4.462978 2.992975 2.335181 2.664078 4930528D03Rik -2.60253 -1.67496 9.169277 3.747484 6.45838 3.523811 4.187894 3.855853 Cacybp -5.62845 -1.67475 15.73031 12.20965 13.96998 7.623814 9.059255 8.341534 Dcun1d1 -8.12154 -1.6747 25.58216 14.73526 20.15871 11.41427 12.66007 12.03717 Cep85l -28.9039 -1.67373 79.40899 64.20124 71.80511 38.80296 46.99952 42.90124 Dnajc9 -5.03431 -1.67311 14.79875 10.22832 12.51354 7.791342 7.16711 7.479226 Kifap3 -29.6946 -1.67256 78.62797 69.0647 73.84633 40.41445 47.88898 44.15172 Gm26708 -3.66716 -1.67163 9.718091 8.536373 9.127232 5.037917 5.882226 5.460071 4921507P07Rik -40.0107 -1.67017 118.0773 81.34866 99.713 55.27862 64.12598 59.7023 Ccpg1os -24.2208 -1.66823 79.95388 40.98037 60.46713 31.69922 40.79347 36.24635 RP23-28J6.1 -1.50779 -1.66811 4.565609 2.963591 3.7646 1.727432 2.786183 2.256807 Sgms2 -4.95095 -1.66675 13.91614 10.83679 12.37646 7.562904 7.288117 7.42551 Yipf1 -1.26686 -1.66595 3.407291 2.931114 3.169202 1.705556 2.099131 1.902343 4930453H23Rik -7.12015 -1.66586 21.46835 14.15823 17.81329 9.728787 11.6575 10.69314 Tspan6 -8.92143 -1.66565 26.51 18.13785 22.32392 11.87198 14.933 13.40249 Tmem184c -4.10789 -1.66459 14.03187 6.546094 10.28898 5.261149 7.101031 6.18109 RP24-181E10.3 -2.64878 -1.66423 6.850209 6.422797 6.636503 3.864178 4.111272 3.987725 Mlf1 -36.9021 -1.66372 104.3058 80.69527 92.50054 51.88687 59.31008 55.59847 Cep19 -8.38614 -1.66284 23.15168 18.92418 21.03793 11.39957 13.90401 12.65179 Lrif1 -3.04483 -1.66276 9.251754 6.026276 7.639015 3.607671 5.580695 4.594183 Pmis2 -14.503 -1.66272 46.12945 26.645 36.38722 21.00967 22.75872 21.88419 Ubtd2 -11.1666 -1.66236 33.24344 22.80742 28.02543 16.34779 17.36984 16.85881 Gm13850 -1.35231 -1.66203 4.038825 2.751113 3.394969 1.828083 2.257243 2.042663 Cst13 -13.7293 -1.66179 40.34756 28.60221 34.47488 18.14333 23.34791 20.74562 Cep112 -3.82771 -1.661 11.81692 7.42015 9.618533 5.503975 6.077672 5.790824 Sypl -4.71887 -1.66029 12.94919 10.78181 11.8655 6.419788 7.873483 7.146635 Als2cr11_2 -31.9307 -1.65947 92.47333 68.22595 80.34964 44.95641 51.88145 48.41893 Ccdc183 -8.23485 -1.6594 26.16135 15.28525 20.7233 13.07464 11.90226 12.48845 Cmtm1 -9.08155 -1.65749 25.53048 20.25767 22.89407 13.63811 13.98694 13.81252

102

Erich3 -26.8534 -1.65748 87.4865 47.90615 67.69633 40.77679 40.90899 40.84289 Minos1 -5.20081 -1.65741 15.56572 10.65793 13.11182 7.909901 7.912132 7.911017 Slc16a7 -28.8082 -1.65733 82.29178 62.97678 72.63428 38.60011 49.05198 43.82605 Rangrf -53.4883 -1.65499 147.7562 122.5468 135.1515 74.39595 88.9303 81.66313 Zp3r -11.1075 -1.65398 32.42612 23.75764 28.09188 15.48711 18.48164 16.98437 Znrd1as -19.7326 -1.65393 66.12531 33.69076 49.90803 29.26574 31.08512 30.17543 Dcdc2c -3.92503 -1.65325 10.12282 9.744193 9.933509 4.940422 7.076534 6.008478 Ccdc88a -5.22052 -1.6524 13.84491 12.60029 13.2226 7.110909 8.893244 8.002077 Gm26799 -3.12863 -1.6522 12.32519 3.526115 7.925653 4.492338 5.101705 4.797021 Cox7a2 -20.2156 -1.65206 58.33779 44.09882 51.2183 29.01696 32.98842 31.00269 Ndufaf5 -1.12272 -1.65115 2.500404 3.193486 2.846945 1.876325 1.572121 1.724223 Tex30 -6.21668 -1.65103 16.40298 15.12827 15.76562 7.780618 11.31727 9.548945 Tmem217 -8.94681 -1.6495 25.97834 19.46514 22.72174 15.98008 11.56977 13.77493 Prss52 -16.1352 -1.64938 56.40577 25.55881 40.98229 27.8865 21.80764 24.84707 Hmox2 -89.9781 -1.64867 236.9947 220.3826 228.6887 147.1104 130.3108 138.7106 4930578I06Rik -54.6795 -1.64842 153.9069 124.1074 139.0072 87.91089 80.74437 84.32763 Arhgap18 -1.04325 -1.64799 2.42338 2.88305 2.653215 1.635901 1.584036 1.609969 Dpf3 -3.026 -1.64792 8.712639 6.679986 7.696312 4.327166 5.01345 4.670308 D6Ertd474e -1.32427 -1.64763 3.231453 3.506674 3.369064 2.197949 1.891644 2.044796 Rbm28 -6.71684 -1.64754 19.90161 14.27782 17.08971 9.701148 11.04459 10.37287 4933417A18Rik -12.857 -1.64705 41.66611 23.78852 32.72732 17.54272 22.19783 19.87027 Gm11635 -2.14454 -1.64616 6.012397 4.914534 5.463465 3.330105 3.307741 3.318923 Gad1os -2.50915 -1.64553 6.457878 6.334405 6.396141 4.025947 3.748034 3.886991 Gm17202 -2.38702 -1.64452 6.173612 6.007514 6.090563 3.34061 4.066479 3.703544 Gm16279 -1.23533 -1.64436 3.860591 2.444334 3.152463 2.401746 1.432525 1.917136 Mbd4 -1.46407 -1.64434 4.310528 3.162049 3.736289 1.990559 2.553871 2.272215 Spata4 -117.363 -1.64393 330.1841 269.0638 299.6239 169.4478 195.074 182.2609 Zkscan7 -1.0309 -1.64333 3.4717 1.794974 2.633337 2.179216 1.025663 1.602439 Psd -1.63787 -1.6433 4.94255 3.425253 4.183902 2.795138 2.296931 2.546034 Itgae -5.20099 -1.64302 12.51106 14.06773 13.2894 7.81168 8.365127 8.088403 Prss54 -16.8674 -1.64288 46.27294 39.93655 43.10475 26.02193 26.45275 26.23734 Fam185a -1.09455 -1.64223 3.015462 2.582213 2.798838 1.354619 2.053959 1.704289 A630095N17Rik -1.05584 -1.64165 2.65552 2.747194 2.701357 1.334414 1.956621 1.645517 Gm4868 -5.52056 -1.64143 15.41436 12.84015 14.12725 8.779367 8.434023 8.606695 1700001C02Rik -9.99152 -1.6409 29.80712 21.3555 25.58131 15.9383 15.24128 15.58979 Ccdc178 -7.61258 -1.63942 22.34998 16.68601 19.518 11.33101 12.47982 11.90541 4921504E06Rik -4.77265 -1.63904 13.81829 10.66387 12.24108 7.424115 7.51274 7.468427 Rgs10 -3.42624 -1.63833 9.640567 7.946982 8.793775 5.29002 5.445054 5.367537 Gar1 -2.785 -1.63819 7.231363 7.066435 7.148899 4.266575 4.461232 4.363903 Fank1 -50.8639 -1.63812 132.9884 128.1583 130.5734 75.47406 83.94492 79.70949 Zdhhc4 -8.5578 -1.63549 23.1028 20.94554 22.02417 14.04365 12.8891 13.46637 Ncbp2 -4.55071 -1.63513 12.12913 11.30236 11.71575 5.90317 8.426897 7.165034 Ccdc83 -5.2472 -1.63446 17.30178 9.733353 13.51757 7.550015 8.990711 8.270363

103

Ccdc117 -10.319 -1.63425 31.72734 21.44971 26.58852 14.7366 17.80244 16.26952 Galntl6_1 -2.56741 -1.63368 8.222836 5.01516 6.618998 3.688731 4.414455 4.051593 Ctag2 -7.05013 -1.63368 24.44384 11.90796 18.1759 10.1073 12.14424 11.12577 4933402J07Rik -55.3893 -1.63356 181.8819 103.7479 142.8149 91.47573 83.3755 87.42561 1700030C14Rik -6.34258 -1.63311 19.72321 12.99816 16.36068 10.38675 9.649452 10.0181 Chpt1 -3.23337 -1.63277 9.191585 7.4949 8.343243 4.644704 5.575043 5.109873 Tiprl -9.64254 -1.63158 26.97927 22.84047 24.90987 14.96958 15.56507 15.26733 Tmem255a -1.76246 -1.63127 4.171853 4.936972 4.554413 2.256164 3.327737 2.79195 Tpte -1.04319 -1.63083 3.107624 2.286155 2.696889 1.410951 1.89644 1.653696 Daf2 -21.1563 -1.63006 62.5361 46.93307 54.73458 32.31494 34.84172 33.57833 Hnrnph1 -5.74381 -1.62971 20.49844 9.231786 14.86511 8.403355 9.839263 9.121309 Dnajc15 -39.3135 -1.62934 127.5639 75.99894 101.7814 61.05192 63.88402 62.46797 Ptch2 -1.7338 -1.6292 5.038678 3.9401 4.489389 2.789395 2.721779 2.755587 9330104G04Rik -1.04256 -1.62807 3.002163 2.402844 2.702504 1.904194 1.4157 1.659947 Hist2h2aa2 -2.67865 -1.62757 6.679301 7.214553 6.946927 3.887016 4.649545 4.268281 RP23-46B12.8 -1.55231 -1.62711 4.102799 3.952491 4.027645 3.015944 1.934716 2.47533 Ttc24 -9.17947 -1.62691 27.66828 19.97535 23.82182 14.96635 14.31833 14.64234 Zfp51 -1.36378 -1.62629 4.050274 3.032362 3.541318 1.752147 2.602937 2.177542 Haglr -3.9195 -1.62571 10.30793 10.05926 10.1836 7.171376 5.356812 6.264094 Gm3448 -47.97 -1.62566 168.5936 80.68835 124.641 78.81048 74.53148 76.67098 Hspa13 -1.58966 -1.625 4.253255 4.012966 4.133111 2.074677 3.012226 2.543451 Glcci1 -2.47841 -1.62491 7.305418 5.583502 6.44446 4.521145 3.410948 3.966046 Emc2 -4.70643 -1.62485 13.92268 10.55441 12.23854 6.451576 8.612651 7.532113 Mrpl30 -1.72763 -1.6244 5.158935 3.830042 4.494489 2.587638 2.94608 2.766859 Gm11691 -1.66063 -1.62436 3.257476 5.383207 4.320342 2.915107 2.404324 2.659715 RP23-441D9.9 -2.04583 -1.62396 6.01691 4.63228 5.324595 4.050127 2.507412 3.27877 Isg20 -7.57795 -1.62393 22.86423 16.58251 19.72337 12.11312 12.17772 12.14542 Cacul1 -7.2876 -1.62324 20.02263 17.93869 18.98066 10.98417 12.40195 11.69306 Sclt1 -5.28266 -1.62313 17.0861 10.43438 13.76024 7.653234 9.301922 8.477578 Rasef -1.86885 -1.62298 5.887436 3.849913 4.868675 3.183143 2.816516 2.99983 Galm -3.49078 -1.62249 9.936481 8.260526 9.098503 5.713362 5.502087 5.607724 Glipr1l1 -4.40246 -1.62228 14.66528 8.289162 11.47722 6.890641 7.258888 7.074764 Zfp330 -7.62484 -1.62139 26.76145 13.02938 19.89541 11.73787 12.80328 12.27058 Dnaja4 -22.0632 -1.62129 64.08421 51.06651 57.57536 34.75594 36.26836 35.51215 Prss46 -11.3883 -1.62123 33.71811 25.72189 29.72 16.87814 19.78536 18.33175 Ccdc54 -45.9545 -1.62119 149.4957 90.36997 119.9329 60.4044 87.55231 73.97835 1700074P13Rik -19.4162 -1.62024 58.62413 42.81761 50.72087 28.7682 33.84105 31.30463 Ttll9 -17.0371 -1.61981 50.4268 38.62224 44.52452 28.26249 26.71234 27.48741 Gm16322 -1.22217 -1.61901 4.009571 2.383581 3.196576 2.344533 1.604276 1.974404 Gm5460 -1.25364 -1.61873 3.917694 2.641917 3.279806 2.204904 1.847428 2.026166 Pgk2 -173.719 -1.61862 482.2342 426.8351 454.5347 252.0797 309.5522 280.8159 Aldh3b2 -4.86154 -1.61854 13.75466 11.6878 12.72123 8.371146 7.34823 7.859688 Lrrc34 -35.2667 -1.61828 91.86153 92.75176 92.30665 54.67993 59.3999 57.03991

104

Rnf146 -4.41435 -1.61823 12.61672 10.49249 11.5546 6.742452 7.538059 7.140256 Gm13225 -1.87761 -1.61809 6.074274 3.756499 4.915387 2.758903 3.316656 3.03778 Cdc16 -2.7171 -1.61799 7.267966 6.959617 7.113792 4.346414 4.44697 4.396692 Pex3 -4.87317 -1.61758 15.35508 10.17277 12.76393 6.955282 8.826225 7.890753 Cib4 -6.60294 -1.6175 21.81351 12.77843 17.29597 10.7568 10.62926 10.69303 Bola1 -2.64778 -1.61733 8.699082 5.174568 6.936825 3.379348 5.198752 4.28905 Selk -9.35496 -1.61732 27.73218 21.28608 24.50913 14.50207 15.80628 15.15417 4933434E20Rik -5.34411 -1.61668 17.25679 10.76341 14.0101 7.86814 9.463841 8.665991 Gm8871 -6.65015 -1.61463 19.22522 15.7147 17.46996 10.28001 11.35962 10.81981 Ankrd45 -4.17681 -1.61458 12.24538 9.700648 10.97302 6.393119 7.199295 6.796207 Lrrc72 -1.75775 -1.61447 4.278751 4.957927 4.618339 2.661395 3.059791 2.860593 Gm20611 -3.35529 -1.61413 10.46455 7.173003 8.818775 5.564896 5.362075 5.463485 2610528A11Rik -3.1163 -1.61358 9.904479 6.485938 8.195209 4.85037 5.307451 5.078911 Dhrs13 -1.24194 -1.61321 2.700535 3.833936 3.267236 2.620743 1.429855 2.025299 Wsb1 -1.10536 -1.6121 2.641262 3.181135 2.911198 1.473365 2.138316 1.80584 Dgkh -14.688 -1.61145 40.14756 37.27154 38.70955 24.73528 23.30773 24.0215 Csde1 -44.7226 -1.61119 125.0918 110.6998 117.8958 68.56004 77.78629 73.17317 4921517D22Rik -2.398 -1.61117 6.970132 5.673143 6.321637 3.147807 4.699476 3.923642 Gm11419 -2.46657 -1.61061 6.822265 6.189938 6.506102 4.475895 3.603161 4.039528 Cep57 -11.627 -1.61051 36.74178 24.60206 30.67192 17.9252 20.16456 19.04488 Usp12 -9.3459 -1.61022 25.87443 23.44865 24.66154 14.67859 15.9527 15.31564 Gm6432 -3.47304 -1.60979 9.459353 8.87757 9.168461 5.333175 6.057678 5.695426 A330021E22Rik -14.0554 -1.6094 38.28785 35.95188 37.11987 19.61814 26.51076 23.06445 Uqcrq -8.77591 -1.60939 25.44843 20.90556 23.17699 12.14552 16.65664 14.40108 Rpl22 -3.88628 -1.60916 12.48538 8.046684 10.26603 6.177453 6.582042 6.379747 Dnajc18 -5.50685 -1.60882 17.34394 11.76008 14.55201 9.010417 9.0799 9.045158 1700007K09Rik -2.61011 -1.60851 8.085789 5.713146 6.899467 4.054892 4.523822 4.289357 Cdk19os -5.92129 -1.60794 16.58223 14.74032 15.66128 8.532652 10.94733 9.73999 Mterfd3 -1.62933 -1.60693 4.466077 4.161652 4.313865 2.336393 3.032674 2.684534 Arl14ep -1.52616 -1.60687 4.490342 3.59155 4.040946 2.233859 2.795717 2.514788 Ppp4r2 -4.50936 -1.60648 10.23808 13.6513 11.94469 7.194343 7.676315 7.435329 Egr3 -1.19317 -1.60623 4.870211 1.452495 3.161353 2.07426 1.862106 1.968183 Gata2 -2.40226 -1.60583 6.563219 6.171766 6.367492 4.31095 3.61952 3.965235 Rps23 -10.5968 -1.60575 30.94078 25.24062 28.0907 16.96768 18.02006 17.49387 Fhl5 -28.3189 -1.60573 90.32714 59.81483 75.07099 40.43902 53.06509 46.75205 Sh3rf2 -7.42629 -1.60572 21.07228 18.30101 19.68665 11.48688 13.03383 12.26036 RP23-11P15.2 -1.24602 -1.60489 4.940717 1.671121 3.305919 2.191659 1.928148 2.059903 Bag5 -86.4923 -1.60483 266.2739 192.7162 229.495 143.7601 142.2453 143.0027 Zbbx -33.9761 -1.60461 97.3388 83.00444 90.17162 54.19268 58.19836 56.19552 Gstk1 -1.22445 -1.6038 2.204545 4.300217 3.252381 2.449238 1.606617 2.027928 Grcc10 -2.13184 -1.60281 5.531526 5.805108 5.668317 4.55293 2.520025 3.536477 Serp2 -3.37996 -1.60209 11.84749 6.139947 8.993721 5.813498 5.414018 5.613758 Sox17 -1.03724 -1.60137 2.806992 2.717095 2.762044 1.805478 1.644132 1.724805

105

Rpain -3.34232 -1.60071 11.01779 6.794663 8.906227 5.715781 5.412032 5.563906 Vti1b -4.93644 -1.60069 14.68412 11.62476 13.15444 8.366578 8.069433 8.218006 Fam71f1 -70.2621 -1.60067 215.3455 159.1243 187.2349 109.9242 124.0215 116.9729 Clip1 -11.2451 -1.60066 35.12415 24.80884 29.9665 19.14887 18.29389 18.72138 Emp3 -1.82873 -1.60037 5.752602 3.996851 4.874726 2.836756 3.255237 3.045996 Atg3 -2.37547 -1.59948 7.386822 5.289279 6.33805 3.382896 4.542263 3.96258 Akip1 -2.85467 -1.59921 9.283509 5.95393 7.618719 4.409518 5.118579 4.764049 Meig1 -78.7413 -1.59858 278.7308 141.8436 210.2872 127.8252 135.2666 131.5459 Pgrmc2 -5.37165 -1.59853 16.03986 12.6528 14.34633 8.948757 9.000599 8.974678 Ankrd42 -29.8402 -1.59838 91.67519 67.74206 79.70863 46.1643 53.57256 49.86843 Spam1 -6.98494 -1.59803 20.92541 16.40436 18.66489 10.39231 12.96758 11.67995 4921504A21Rik -1.59485 -1.59742 4.236007 4.292809 4.264408 2.3632 2.975915 2.669557 Ccnd3 -6.23628 -1.59741 18.95356 14.39664 16.6751 10.44671 10.43094 10.43882 Gm14340 -1.37502 -1.59739 4.725373 2.628107 3.67674 2.44405 2.159381 2.301715 Gm6309 -2.18502 -1.59706 9.609935 2.079308 5.844622 2.597135 4.722068 3.659602 Izumo1 -10.0505 -1.59645 32.7979 21.00421 26.90106 17.75017 15.95088 16.85052 RP24-93I18.4 -7.16078 -1.59614 20.19855 18.14678 19.17267 14.28914 9.734629 12.01188 Fam161a -11.8287 -1.59569 35.16965 28.20183 31.68574 19.58065 20.13345 19.85705 Tfam -4.49892 -1.5955 16.35797 7.749693 12.05383 7.009878 8.099951 7.554914 Smad6 -1.74962 -1.59505 5.03034 4.349478 4.689909 2.973716 2.906861 2.940288 Gm26787 -1.35267 -1.59452 4.315931 2.939869 3.6279 1.233795 3.316656 2.275226 Pabpn1 -3.54422 -1.59448 12.98856 6.023745 9.506152 6.971824 4.952036 5.96193 Spata24 -94.6719 -1.59444 319.2819 188.587 253.9344 179.8256 138.6993 159.2625 Ift74 -13.1713 -1.59428 46.10485 24.56481 35.33483 19.9054 24.42163 22.16351 Clhc1 -1.92233 -1.59422 5.212371 5.102403 5.157387 3.298561 3.171543 3.235052 3110082I17Rik -1.33286 -1.5929 4.043326 3.118442 3.580884 2.097337 2.398715 2.248026 Rnasek -1.48209 -1.59258 4.738584 3.227766 3.983175 1.625543 3.376619 2.501081 BC029722 -1.47385 -1.59246 4.385513 3.537552 3.961532 2.635216 2.340142 2.487679 Gm6880 -2.58701 -1.59221 6.985695 6.925025 6.95536 4.110847 4.625863 4.368355 A630023A22Rik -1.10467 -1.59207 2.938491 3.002404 2.970447 1.620051 2.111505 1.865778 Gm4922 -2.66158 -1.59206 8.220025 6.094022 7.157024 3.627252 5.363629 4.495441 1600016N20Rik -24.0598 -1.59192 72.03453 57.37878 64.70665 40.62439 40.66932 40.64686 1700010D01Rik -5.20102 -1.59109 21.68969 6.310378 14.00003 9.1036 8.494435 8.799017 1700024J04Rik -1.21238 -1.59095 4.708225 1.819673 3.263949 2.172523 1.930618 2.051571 Ccdc173 -3.86631 -1.58998 14.31764 6.521652 10.41964 6.303505 6.803165 6.553335 Lrrfip2 -18.0608 -1.58957 60.83217 36.55712 48.69465 31.77288 29.49484 30.63386 Pkib -1.31358 -1.58899 4.327906 2.75974 3.543823 2.158924 2.301563 2.230243 Ginm1 -2.10848 -1.58888 5.622821 5.75511 5.688966 3.333599 3.82738 3.580489 Catip -3.21281 -1.5885 9.230701 8.113649 8.672175 5.313708 5.605014 5.459361 Eif1ax -3.28575 -1.58846 10.12266 7.616081 8.869369 5.258859 5.908385 5.583622 Nr4a1 -1.10675 -1.58822 3.390175 2.586388 2.988281 1.986756 1.776301 1.881529 Exosc3 -7.40355 -1.58818 22.03107 17.95067 19.99087 12.49544 12.6792 12.58732 Ccdc12 -5.0465 -1.58802 14.12693 13.13046 13.62869 8.353759 8.810629 8.582194

106

Lgals8 -14.8494 -1.58782 42.5783 37.64356 40.11093 24.02226 26.50088 25.26157 4930544G11Rik -43.7539 -1.58777 123.6315 112.7574 118.1944 72.20461 76.6764 74.44051 Gm16252 -2.00011 -1.58726 7.611961 3.199895 5.405928 3.427249 3.384383 3.405816 Zfp131 -2.73203 -1.58717 8.659851 6.109918 7.384884 4.749226 4.556483 4.652854 Cldn25 -3.21192 -1.58682 8.944701 8.425943 8.685322 4.911352 6.035461 5.473406 Mfap3l -12.5431 -1.58681 37.77028 30.06567 33.91798 20.4716 22.27816 21.37488 Gm11193 -4.30647 -1.58664 14.6097 8.685067 11.64738 6.12233 8.559495 7.340912 RP24-89G21.3 -5.4655 -1.58663 14.65285 14.91167 14.78226 9.236328 9.397193 9.316761 Bud31 -5.72251 -1.58659 18.24458 12.71166 15.47812 10.17038 9.340851 9.755613 Adam30 -8.62969 -1.58651 25.01432 21.67222 23.34327 14.05919 15.36797 14.71358 Actg2 -38.6798 -1.58635 110.6135 98.68161 104.6475 61.93739 69.99801 65.9677 Dydc1 -4.82177 -1.58587 15.07933 11.02436 13.05185 7.891846 8.568307 8.230076 Rnf125 -5.23413 -1.58563 14.81015 13.53339 14.17177 8.717185 9.158099 8.937642 Wbscr28 -9.21915 -1.58555 25.89782 24.02954 24.96368 16.40041 15.08865 15.74453 Fam122c -3.66854 -1.58543 12.17586 7.694086 9.934972 6.084553 6.448313 6.266433 Zfp759 -1.16049 -1.58477 4.255471 2.034549 3.14501 1.394398 2.574647 1.984523 4930483J18Rik -3.17032 -1.5843 10.11755 7.074803 8.596178 6.30432 4.547392 5.425856 Golga5 -9.2153 -1.58392 28.34045 21.65355 24.997 15.78725 15.77616 15.7817 Celf5 -1.76412 -1.58321 6.219332 3.358658 4.788995 3.110362 2.939379 3.02487 1700110K17Rik -1.14462 -1.58314 2.991577 3.22337 3.107473 2.215751 1.709949 1.96285 Hist1h2ap -1.0465 -1.58306 2.126792 3.5559 2.841346 2.860297 0.729389 1.794843 Gm21586 -1.10735 -1.58302 2.118003 3.895327 3.006665 1.135263 2.663376 1.899319 Gm13817 -2.606 -1.58274 10.18325 3.972717 7.077982 5.160559 3.783404 4.471982 4933436I01Rik -1.7137 -1.58138 7.319439 2.003207 4.661323 2.690239 3.205016 2.947627 Rpl22l1 -2.10028 -1.5796 7.241925 4.205997 5.723961 2.574191 4.673162 3.623677 1700095B10Rik -3.56396 -1.57937 11.79139 7.639433 9.715409 6.679361 5.623533 6.151447 1700003H04Rik -7.7676 -1.57921 21.62203 20.73468 21.17836 11.4989 15.32261 13.41075 Zdhhc25 -13.4416 -1.57893 38.48945 34.83014 36.65979 20.28735 26.14898 23.21816 Eml6 -1.18898 -1.57881 4.208159 2.278116 3.243138 1.711887 2.396435 2.054161 Zfp711 -1.17991 -1.57861 3.754625 2.683682 3.219153 1.537804 2.540673 2.039239 Maats1 -5.34416 -1.57813 15.97051 13.20551 14.58801 8.950299 9.537399 9.243849 Tomm7 -6.9288 -1.57788 17.72722 20.11027 18.91874 9.057885 14.92201 11.98995 Ap4m1 -4.21605 -1.57783 12.28907 10.73569 11.51238 7.179852 7.412818 7.296335 Ngly1 -18.0419 -1.57781 52.99265 45.54051 49.26658 29.40266 33.04676 31.22471 4930440I19Rik -1.13195 -1.57775 3.655897 2.526498 3.091198 1.4109 2.5076 1.95925 4930567H17Rik -2.17337 -1.57763 7.655069 4.216846 5.935957 4.281563 3.243611 3.762587 Erich2 -11.8574 -1.57759 40.43173 24.34095 32.38634 20.96339 20.09457 20.52898 Usp50 -9.90665 -1.57711 28.32222 25.82275 27.07249 18.33334 15.99834 17.16584 Eed -2.53501 -1.57696 7.520917 6.336492 6.928704 4.269981 4.517412 4.393696 Atp6v1c1 -10.0535 -1.57666 29.41974 25.55522 27.48748 15.37851 19.48951 17.43401 Psma6 -37.4523 -1.57662 115.5462 89.26211 102.4041 58.67893 71.22479 64.95186 Fbxw10 -21.4162 -1.57632 62.67606 54.47715 58.57661 36.45658 37.86427 37.16042 Fsip1 -14.9761 -1.57597 44.11881 37.83693 40.97787 26.19233 25.81114 26.00174

107

Plk2 -1.46972 -1.5758 3.558729 4.485696 4.022213 2.438749 2.666227 2.552488 Adh4 -2.43072 -1.57525 3.856227 9.456241 6.656234 2.935085 5.515943 4.225514 Vps26a -28.3732 -1.57511 87.91928 67.49664 77.70796 43.83204 54.83753 49.33478 Fam109a -2.01399 -1.57497 5.308161 5.725379 5.51677 3.542604 3.462959 3.502781 Prps1 -2.19153 -1.57459 5.704687 6.306533 6.00561 3.218154 4.410005 3.81408 Coro2a -5.56225 -1.57445 17.32834 13.16173 15.24504 9.772225 9.593348 9.682787 Tmem203 -2.52187 -1.57433 5.128842 8.696826 6.912834 4.562321 4.21961 4.390966 Ifngr1 -1.21329 -1.57385 3.404279 3.250894 3.327587 2.252821 1.975781 2.114301 Csnka2ip -12.6896 -1.57253 43.10834 26.59863 34.85349 19.92081 24.40697 22.16389 Nsmce4a -1.0311 -1.57133 2.547802 3.123861 2.835832 1.984983 1.624485 1.804734 Igsf11 -5.48698 -1.57114 16.83637 13.35172 15.09404 9.405246 9.808874 9.60706 Ptpn2 -2.04065 -1.57104 6.292334 4.936158 5.614246 3.069024 4.078161 3.573593 Gm15492 -3.52311 -1.57089 10.14964 9.23908 9.69436 4.9457 7.396801 6.17125 Slc25a4 -2.20047 -1.56998 5.663611 6.45855 6.06108 3.948573 3.772644 3.860608 Flywch2 -1.00901 -1.56969 2.806992 2.753323 2.780158 1.865661 1.676643 1.771152 Morn3 -19.8296 -1.56911 64.3322 45.01266 54.67243 36.81575 32.86995 34.84285 Dnajb4 -7.43274 -1.56906 23.17933 17.80893 20.49413 11.81347 14.30932 13.06139 Tfb1m -4.84106 -1.56894 17.4867 9.213362 13.35003 7.679066 9.338875 8.50897 1700029N11Rik -1.32663 -1.56876 4.779084 2.539176 3.65913 2.561619 2.103384 2.332501 Lamtor3 -1.61094 -1.56845 4.389746 4.499929 4.444838 2.545794 3.122011 2.833903 Exosc4 -3.3 -1.56842 8.576552 9.634508 9.10553 6.595148 5.01591 5.805529 Ppp1r11 -90.5335 -1.56816 259.9471 239.8075 249.8773 163.3758 155.3119 159.3438 Sqle -9.22369 -1.56806 25.00359 25.91827 25.46093 14.91522 17.55926 16.23724 Gm12853 -1.37606 -1.56797 5.271049 2.32662 3.798835 3.254763 1.590794 2.422779 Gm8251 -3.28378 -1.56718 11.10209 7.044781 9.073435 4.897507 6.681802 5.789655 Cox6a1 -24.0873 -1.56707 65.57966 67.54859 66.56412 38.25351 46.70013 42.47682 Meiob -1.3676 -1.56676 4.541954 3.019278 3.780616 1.93588 2.890146 2.413013 Dlgap4 -4.50751 -1.56674 13.32104 11.60071 12.46087 7.929834 7.97689 7.953362 Gm1698 -5.10942 -1.56664 16.84147 11.41147 14.12647 7.204429 10.82968 9.017054 Gm16405 -1.59941 -1.56621 5.435104 3.413259 4.424181 2.899038 2.750513 2.824776 Alas2 -3.24331 -1.56546 9.113672 8.84432 8.978996 5.911871 5.559506 5.735688 Wfdc15a -5.00949 -1.56468 11.83999 15.92188 13.88093 9.458043 8.284845 8.871444 Iqcf5 -6.02545 -1.56392 19.112 14.30879 16.71039 9.246933 12.12295 10.68494 Ift81 -10.836 -1.56391 35.51719 24.58655 30.05187 17.96694 20.46472 19.21583 Rbm43 -3.09759 -1.56372 9.981081 7.203815 8.592448 5.281624 5.708101 5.494862 Spesp1 -25.1762 -1.56316 76.58273 63.18022 69.88147 43.34994 46.06058 44.70526 Sema4c -1.29438 -1.56298 4.6045 2.582552 3.593526 2.126716 2.471585 2.29915 1700011L22Rik -26.2726 -1.56265 87.28426 58.65029 72.96727 44.68061 48.70873 46.69467 Mrpl18 -2.08714 -1.5626 6.199478 5.394386 5.796932 3.773166 3.646424 3.709795 Hist1h4a -2.5754 -1.56218 7.953319 6.359717 7.156518 3.781121 5.381106 4.581113 BC048671 -9.10644 -1.56201 28.28251 22.33711 25.30981 14.50405 17.90269 16.20337 Swi5 -17.3223 -1.5616 55.77346 40.55975 48.1666 31.47351 30.21513 30.84432 Gdpd1 -13.2608 -1.56012 39.97369 33.89735 36.93552 20.86162 26.48782 23.67472

108

Kif7 -1.05003 -1.55968 2.909076 2.943249 2.926162 1.910394 1.841863 1.876128 C2cd4b -1.04838 -1.55933 3.090018 2.755398 2.922708 1.761668 1.986995 1.874331 2810029C07Rik -2.20279 -1.5589 7.073045 5.215077 6.144061 3.78095 4.101598 3.941274 Pagr1b -1.12459 -1.55887 2.151622 4.122039 3.136831 2.248902 1.775584 2.012243 4921524L21Rik -4.97958 -1.5586 15.04927 12.73877 13.89402 8.505267 9.323614 8.914441 4931409K22Rik -6.99101 -1.55807 27.22467 11.81145 19.51806 12.84148 12.21262 12.52705 Rpl26 -25.2632 -1.55791 92.03047 49.06025 70.54536 43.08842 47.47596 45.28219 Dnajc5g -21.0441 -1.5574 66.85895 50.73693 58.79794 35.78182 39.72584 37.75383 Tcp10c -64.5262 -1.55709 177.8318 182.8749 180.3534 115.4972 116.1571 115.8271 Gm10338 -2.17588 -1.55671 9.158701 3.009965 6.084333 3.097301 4.719599 3.90845 RP24-92M6.1 -2.31605 -1.5565 9.027989 3.927788 6.477888 2.3727 5.95098 4.16184 Hist1h4f -3.0308 -1.55642 6.435263 10.52037 8.477817 7.97181 2.92222 5.447015 Gm20605 -3.35779 -1.5563 10.41731 8.370066 9.393689 5.948534 6.123258 6.035896 Zfp654 -9.36147 -1.55621 25.63226 26.75213 26.19219 14.36279 19.29866 16.83072 Kcnu1 -9.15233 -1.55589 27.81247 23.42093 25.6167 15.51379 17.41494 16.46437 Ccdc39 -8.178 -1.55564 27.80552 17.98688 22.8962 12.66905 16.76736 14.7182 Tipin -4.59439 -1.55522 11.77407 13.96442 12.86924 7.622667 8.927037 8.274852 Igf2bp3 -7.36177 -1.55445 22.18248 19.09643 20.63945 13.03984 13.51553 13.27769 Gm7461 -1.92591 -1.55402 7.211968 3.592304 5.402136 3.551575 3.400876 3.476225 Clip4 -41.792 -1.55384 125.6198 108.8809 117.2503 73.36413 77.55251 75.45832 Hnrnpa1 -4.58102 -1.55376 15.93693 9.770134 12.85353 8.938154 7.606867 8.27251 Rin1 -1.35748 -1.55312 4.887232 2.736181 3.811706 2.698533 2.209913 2.454223 4930517O19Rik -1.2301 -1.55298 3.942745 2.966441 3.454593 2.42073 2.028263 2.224496 Klhl41 -1.13195 -1.55283 3.648091 2.710864 3.179478 1.801338 2.293726 2.047532 Nsun7 -7.7883 -1.5527 23.47615 20.28311 21.87963 12.87582 15.30684 14.09133 Chn2 -11.8098 -1.55247 34.47055 31.90213 33.18634 20.55448 22.19851 21.37649 4932416K20Rik -3.54675 -1.55216 13.47799 6.462253 9.970119 6.021174 6.825556 6.423365 Hsf2 -3.87712 -1.55187 12.44702 9.357927 10.90247 5.574389 8.476323 7.025356 Tmco5b -3.6667 -1.55133 12.50598 8.128647 10.31731 5.911811 7.389414 6.650612 U2af1l4 -1.81913 -1.5513 5.071181 5.166462 5.118822 3.041215 3.558169 3.299692 Lrrc51 -51.9038 -1.5513 159.4279 132.6772 146.0525 91.39899 96.89845 94.14872 Nat6 -5.64531 -1.55114 17.38483 14.39162 15.88822 13.2359 7.249925 10.24291 Carf -1.26708 -1.55098 3.778602 3.354891 3.566746 2.198342 2.400991 2.299666 Gm10376 -2.09468 -1.55072 6.307765 5.488612 5.898188 2.617549 4.989468 3.803509 Rhobtb2 -1.10435 -1.55025 3.966384 2.256342 3.111363 2.160482 1.853538 2.00701 Upf3a -43.2546 -1.55011 154.2789 89.48693 121.8829 81.2764 75.98029 78.62835 Timm10 -3.10608 -1.54995 9.376388 8.131594 8.753991 6.766446 4.529373 5.647909 Etf1 -8.056 -1.54987 23.41738 21.99615 22.70677 13.89863 15.40291 14.65077 Psd3 -1.98275 -1.54975 6.661801 4.517058 5.58943 3.325317 3.888034 3.606676 Ccdc37 -6.07888 -1.54922 22.19782 12.09633 17.14708 10.825 11.3114 11.0682 Lrrcc1 -4.8115 -1.54884 15.30969 11.84666 13.57817 7.938791 9.594566 8.766678 Gm13538 -1.03591 -1.54857 2.720939 3.127635 2.924287 1.531362 2.245401 1.888381 Ngfrap1 -1.36462 -1.54835 3.831126 3.87531 3.853218 3.080261 1.896937 2.488599

109

Trp53rk -1.27891 -1.54775 4.38837 2.8392 3.613785 2.333098 2.336644 2.334871 Polb -26.6404 -1.54773 82.48302 68.0735 75.27826 39.32617 57.94958 48.63788 Gm28048 -1.04199 -1.54749 3.393742 2.496641 2.945191 1.789962 2.016445 1.903203 Ccdc23 -14.8976 -1.54717 40.68953 43.55938 42.12446 31.18104 23.27265 27.22684 RP23-362B7.1 -1.33881 -1.54714 4.190889 3.38056 3.785724 1.773428 3.120405 2.446917 Shfm1 -7.26414 -1.547 23.23473 17.85358 20.54415 11.93137 14.62866 13.28001 Odf3 -12.3871 -1.54687 41.17792 28.89789 35.03791 25.58566 19.71595 22.6508 Sqrdl -22.1599 -1.54617 68.25115 57.21539 62.73327 37.58843 43.55832 40.57338 Fam209 -21.2618 -1.54546 75.30139 45.18132 60.24136 36.15406 41.80508 38.97957 Bzw1 -7.86241 -1.54531 23.45401 21.10733 22.28067 12.46993 16.36659 14.41826 Rfk -1.55433 -1.5453 4.332021 4.477456 4.404738 2.256744 3.444073 2.850409 Gm527 -2.40754 -1.54518 8.726811 4.920413 6.823612 3.605527 5.226624 4.416076 Cmss1 -3.31235 -1.54486 8.216942 10.56625 9.391595 5.543013 6.615481 6.079247 Gpd2 -29.9652 -1.54462 88.66701 81.30481 84.98591 52.39995 57.6414 55.02068 Dusp22 -2.94006 -1.54438 9.142775 7.538861 8.340818 4.952169 5.849341 5.400755 Prm3 -33.7516 -1.54422 126.6049 64.93478 95.76986 53.5091 70.52749 62.01829 1700034O15Rik -51.9666 -1.54389 147.1455 147.8787 147.5121 88.25451 102.8365 95.5455 B3gntl1 -1.33144 -1.54386 3.629143 3.930021 3.779582 2.572832 2.323451 2.448142 1700092C17Rik -3.00099 -1.54344 9.231297 7.815152 8.523225 5.654895 5.389567 5.522231 Scamp1 -6.46924 -1.5431 20.82688 15.93496 18.38092 11.0674 12.75595 11.91168 D930016D06Rik -1.22639 -1.54253 4.236946 2.736836 3.486891 1.967487 2.553509 2.260498 5031410I06Rik -1.72537 -1.54172 4.822966 4.99781 4.910388 2.26158 4.108446 3.185013 Tes -8.06357 -1.54163 25.25714 20.64519 22.95117 14.74657 15.02863 14.8876 Pfn3 -2.58133 -1.54141 9.454276 5.24395 7.349113 5.501912 4.033663 4.767788 Ppp1r36 -29.1181 -1.54136 95.65995 70.15026 82.90511 50.91927 56.65473 53.787 Gtsf1 -24.6026 -1.54122 79.7832 60.33739 70.0603 44.50361 46.41172 45.45766 Atp8b4 -1.56358 -1.54117 1.511224 7.394492 4.452858 2.664084 3.11447 2.889277 Speer4d -1.50688 -1.54088 6.505066 2.08066 4.292863 2.637807 2.934159 2.785983 Rnf138rt1 -1.85024 -1.5407 5.555535 4.988809 5.272172 3.523033 3.320834 3.421933 Atp1b3 -31.5291 -1.54057 88.97919 90.73116 89.85518 54.14591 62.50627 58.32609 Gm27211 -5.84427 -1.53942 19.05196 14.3055 16.67873 11.77939 9.889523 10.83446 Pacsin1 -1.16613 -1.53934 3.967246 2.689365 3.328306 2.18372 2.140621 2.162171 Ndufa5 -9.46401 -1.53924 32.32828 21.7011 27.01469 19.51596 15.58539 17.55068 Prkar2a -16.1577 -1.53915 50.32053 41.93316 46.12685 27.53084 32.40737 29.96911 4833439L19Rik -5.26355 -1.53887 15.93653 14.12601 15.03127 9.855521 9.679912 9.767717 Fxr1 -89.8926 -1.53865 269.3867 244.1675 256.7771 148.0355 185.7335 166.8845 4930426I24Rik -2.67327 -1.5381 9.363104 5.919429 7.641266 6.210619 3.725381 4.968 E530001K10Rik -3.39937 -1.53761 9.319215 10.12574 9.722476 6.286588 6.359621 6.323105 1700020N15Rik -2.11934 -1.5376 7.987898 4.135229 6.061564 3.1969 4.687541 3.942221 Dnajc2 -9.8026 -1.53742 34.52316 21.56204 28.0426 18.39139 18.08861 18.24 Nt5c -4.09546 -1.53638 11.93513 11.52652 11.73083 7.098381 8.172358 7.635369 1700025F22Rik -4.52866 -1.53607 15.16543 10.78755 12.97649 8.488664 8.407003 8.447834 Gm13447 -3.34817 -1.53605 8.115046 11.07323 9.594139 6.566667 5.925263 6.245965

110

Tmem237 -6.88362 -1.53507 19.50443 19.99242 19.74842 12.59557 13.13404 12.86481 Tsga13 -1.7277 -1.53424 5.71731 4.205997 4.961653 3.309675 3.158236 3.233955 RP23-18J12.5 -2.21304 -1.53399 10.59002 2.124724 6.35737 5.201568 3.087094 4.144331 Ccdc58 -1.21829 -1.53398 4.108062 2.891551 3.499807 1.908354 2.654679 2.281516 Bean1 -1.00247 -1.5338 3.027506 2.733401 2.880454 1.575555 2.180413 1.877984 Pvrl3 -10.927 -1.53348 31.12254 31.69637 31.40945 19.38342 21.58155 20.48249 St8sia3os -1.64727 -1.53302 4.944889 4.530541 4.737715 2.900977 3.279912 3.090445 Rgs19 -1.0722 -1.53239 2.841734 3.330533 3.086133 2.096622 1.931246 2.013934 Med21 -1.53093 -1.53231 3.812809 5.001041 4.406925 2.908693 2.843299 2.875996 Adora3_2 -14.7599 -1.53174 55.70819 29.32748 42.51784 25.24219 30.2737 27.75795 Spred3 -1.71548 -1.53154 6.417935 3.467709 4.942822 2.87865 3.576038 3.227344 Srsf2 -2.18688 -1.53127 6.773601 5.832894 6.303247 3.1815 5.05123 4.116365 Cep128 -9.62943 -1.53122 31.50893 24.00399 27.75646 18.1465 18.10755 18.12703 Ccdc28a -4.15745 -1.53097 14.33725 9.63756 11.98741 6.734361 8.925543 7.829952 0610009L18Rik -8.46105 -1.53068 23.33567 25.4738 24.40474 16.26856 15.61883 15.94369 Clgn -43.6139 -1.53044 140.7028 110.9699 125.8363 78.78076 85.66409 82.22242 Poli -2.37419 -1.5292 7.86452 5.856603 6.860561 4.422324 4.550422 4.486373 Ppef1 -1.49321 -1.52911 4.480702 4.149985 4.315343 2.239268 3.404994 2.822131 Gm1527 -2.21087 -1.52902 8.791712 3.988422 6.390067 2.96882 5.389567 4.179193 Efcab3 -9.70924 -1.52871 34.08446 22.06184 28.07315 18.93666 17.79116 18.36391 Ttc39a -14.1395 -1.52867 44.16365 37.60611 40.88488 27.03741 26.45345 26.74543 Srsf6 -3.94617 -1.52865 13.18687 9.634818 11.41085 6.823424 8.105926 7.464675 Bbip1 -1.90246 -1.52863 6.180347 4.822189 5.501268 2.746533 4.451092 3.598812 1700123O20Rik -2.36621 -1.52844 6.610772 7.077163 6.843967 3.744384 5.211136 4.47776 Polr2d -3.52242 -1.52807 9.748132 10.63745 10.19279 5.893867 7.446865 6.670366 Adam24 -8.41621 -1.52791 25.94443 22.77296 24.3587 14.30669 17.57828 15.94248 Rab28 -7.1149 -1.52728 20.96254 20.25443 20.60848 12.75047 14.23669 13.49358 RP24-339K18.2 -1.04723 -1.52708 3.761885 2.306224 3.034054 2.199702 1.773955 1.986828 Eno3 -5.58134 -1.52705 17.95248 14.38964 16.17106 9.858182 11.32126 10.58972 2310010J17Rik -1.51948 -1.52693 4.752593 4.053673 4.403133 2.25945 3.507861 2.883655 Tbc1d23 -2.31693 -1.52674 8.200296 5.230786 6.715541 4.074996 4.722235 4.398616 Zfp105 -5.94891 -1.5264 19.42597 15.07412 17.25004 11.17227 11.43001 11.30114 1700001C19Rik -18.0042 -1.52615 60.21117 44.23469 52.22293 35.06094 33.37648 34.21871 Fam96b -3.65325 -1.52561 12.91686 8.290704 10.60378 7.360306 6.540755 6.95053 Gm17673 -10.8842 -1.52556 35.09276 28.09559 31.59418 21.05266 20.3672 20.70993 Pmfbp1 -60.304 -1.52463 201.0296 149.4699 175.2497 119.9367 109.9548 114.9457 1700016H13Rik -30.7298 -1.52405 94.36304 84.37576 89.3694 54.63578 62.64332 58.63955 Lyrm2 -1.23233 -1.524 3.760385 3.407841 3.584113 2.313546 2.390029 2.351788 Bcas2 -3.83828 -1.52368 11.61187 10.72354 11.1677 6.690552 7.968286 7.329419 Cd59a -2.14818 -1.52334 6.638204 5.867726 6.252965 3.938754 4.270813 4.104784 4930504O13Rik -13.9517 -1.52305 52.11291 29.13808 40.6255 27.73267 25.61498 26.67383 Usmg5 -2.29805 -1.52299 6.012397 7.371801 6.692099 4.921914 3.86619 4.394052 Nudcd2 -1.04858 -1.52285 3.493571 2.614585 3.054078 1.400784 2.610212 2.005498

111

Lyrm1 -3.93814 -1.5226 12.07248 10.87499 11.47373 7.17084 7.90035 7.535595 1700001G01Rik -1.56979 -1.52254 4.931021 4.216906 4.573963 2.452047 3.556299 3.004173 Kiz -25.5607 -1.52223 84.35567 64.65535 74.50551 46.57469 51.31499 48.94484 BC049715 -20.7608 -1.52223 71.14222 49.888 60.51511 39.01574 40.49282 39.75428 Ube2u -9.66693 -1.52217 35.47001 20.88997 28.17999 18.21266 18.81345 18.51305 Prss51 -8.48355 -1.52203 27.62776 21.84141 24.73458 15.70159 16.80048 16.25103 Gm10638 -36.3282 -1.52171 114.199 97.7232 105.9611 63.31982 75.94594 69.63288 Mrpl51 -5.4556 -1.52134 17.54466 14.29563 15.92015 10.92774 10.00134 10.46454 Aamdc -2.25449 -1.52087 6.232044 6.933601 6.582823 4.194076 4.462597 4.328337 Fgfr1op2 -8.05859 -1.52069 27.15666 19.91376 23.53521 14.41772 16.53553 15.47662 4930557F10Rik -4.48721 -1.52043 14.7924 11.42629 13.10935 7.897105 9.34718 8.622143 Sept12 -16.6549 -1.52042 56.90654 40.40897 48.65776 32.78653 31.21919 32.00286 Gykl1 -46.7401 -1.52036 148.124 125.0002 136.5621 85.30423 94.33971 89.82197 Spata6 -37.4504 -1.52008 121.0535 97.86407 109.4588 62.58889 81.42787 72.00838 Arhgef39 -1.30017 -1.51999 4.354773 3.246348 3.800561 2.240818 2.759954 2.500386 Azi2 -4.49104 -1.51966 14.66123 11.60552 13.13337 7.866267 9.418398 8.642332 RP23-226D9.5 -4.17144 -1.51922 13.93199 10.47889 12.20544 9.111769 6.956229 8.033999 Ostc -2.07541 -1.51919 5.989665 6.155941 6.072803 3.342695 4.652097 3.997396 Ube2q2 -2.20765 -1.51839 7.013764 5.918845 6.466305 3.801193 4.716124 4.258658 Thoc7 -10.6744 -1.51828 39.89426 22.64554 31.2699 19.89012 21.30097 20.59554 Ndufs6 -4.69485 -1.51811 15.70488 11.80777 13.75633 8.481443 9.641504 9.061474 Mak -14.9962 -1.51792 45.85106 42.05132 43.95119 29.50856 28.40137 28.95496 Zcchc13 -9.80329 -1.51786 31.35035 26.11724 28.7338 20.60675 17.25426 18.9305 Lyrm7 -1.08883 -1.5174 4.067603 2.31893 3.193267 2.083051 2.125814 2.104432 Wdr77 -1.70742 -1.51734 5.372154 4.643436 5.007795 3.121597 3.479145 3.300371 Cep63 -22.1994 -1.51733 74.37859 55.84312 65.11086 40.06049 45.76251 42.9115 Gm6455 -2.00541 -1.51713 8.352899 3.41383 5.883365 3.739784 4.016127 3.877955 Nt5c1b -135.528 -1.51699 413.8453 381.5055 397.6754 246.104 278.1914 262.1477 B230118H07Rik -2.87888 -1.51682 9.625582 7.272972 8.449277 5.157042 5.983746 5.570394 Cdrt4 -34.1941 -1.51641 124.6429 76.17528 100.4091 68.73004 63.69999 66.21502 Atg101 -2.59819 -1.51635 8.106959 7.153044 7.630002 4.775857 5.287775 5.031816 4930584F24Rik -4.11542 -1.51585 11.74074 12.44596 12.09335 6.843758 9.1121 7.977929 4930539J05Rik -2.02885 -1.51566 7.54253 4.384189 5.963359 3.917065 3.951951 3.934508 Fkbp7 -1.16749 -1.51564 4.016362 2.846955 3.431658 2.22007 2.308259 2.264164 Spag4 -13.83 -1.5154 43.88577 37.44115 40.66346 25.48554 28.18128 26.83341 4933433C11Rik -9.22361 -1.51513 31.28765 22.97008 27.12886 18.3678 17.44271 17.90525 Rnf138 -13.7344 -1.51488 42.13124 38.68773 40.40949 24.43933 28.9109 26.67511 2010107E04Rik -20.6518 -1.51447 68.68133 52.90529 60.79331 36.4578 43.8253 40.14155 Med10 -13.4948 -1.51397 44.10951 35.39234 39.75092 24.26881 28.24352 26.25617 Tex9 -1.21216 -1.51339 4.120453 3.026058 3.573255 2.234705 2.487492 2.361099 Yme1l1 -4.3405 -1.51333 13.8933 11.69879 12.79604 7.64897 9.262122 8.455546 AF366264 -11.3278 -1.51304 36.85934 29.95552 33.40743 19.76383 24.39548 22.07965 Gm13001 -7.29307 -1.51299 23.23783 19.78195 21.50989 13.78508 14.64857 14.21683

112

Ccnl2 -2.34605 -1.51297 8.210373 5.628715 6.919544 3.918141 5.228855 4.573498 Mff -7.07724 -1.51294 21.47292 20.27622 20.87457 12.82136 14.7733 13.79733 4833427G06Rik -4.72767 -1.51229 14.7905 13.12181 13.95615 8.817258 9.639701 9.22848 Tnp2 -607.199 -1.51226 1863.843 1721.228 1792.536 1068.472 1302.201 1185.337 4930505A04Rik -6.56709 -1.51225 25.03951 13.73463 19.38707 11.08017 14.55979 12.81998 Als2cr12 -8.93341 -1.5122 30.54266 22.20657 26.37461 16.66229 18.22012 17.44121 H2-Ab1 -1.50696 -1.51173 5.692862 3.210809 4.451835 3.258905 2.630836 2.94487 Pfdn2 -1.413 -1.51138 4.281931 4.070284 4.176108 2.939845 2.586376 2.76311 Slain2 -19.1788 -1.51137 62.69017 50.67631 56.68324 35.85185 39.15708 37.50447 RP23-472J11.6 -1.04453 -1.51125 3.717481 2.457747 3.087614 1.851939 2.234236 2.043087 Catsper1 -16.1555 -1.51117 56.65685 38.86381 47.76033 29.52014 33.68957 31.60486 BC024978 -1.57627 -1.51092 5.342656 3.980131 4.661393 3.044943 3.125311 3.085127 Hook1 -16.7076 -1.51075 54.42744 44.41116 49.4193 30.69157 34.73179 32.71168 1700012P22Rik -7.8549 -1.5104 29.94778 16.54114 23.24446 15.07778 15.70134 15.38956 Eif2s3y -1.97904 -1.51015 6.718972 4.997801 5.858387 3.428543 4.330146 3.879344 Lhb -3.89059 -1.51011 12.89264 10.14249 11.51756 7.909165 7.344783 7.626974 Polr2i -2.97289 -1.51006 8.458033 9.144804 8.801419 5.638098 6.018965 5.828532 Slc25a46 -1.65397 -1.50991 6.16164 3.633618 4.897629 2.646713 3.840599 3.243656 Creld2 -41.5926 -1.50901 147.5087 99.10046 123.3046 81.78288 81.64105 81.71197 Bccip -1.34326 -1.50828 4.66418 3.307906 3.986043 2.352969 2.932588 2.642779 Snrpe -1.33119 -1.50707 4.41508 3.497844 3.956462 1.791266 3.459282 2.625274 Cystm1 -3.9373 -1.50703 12.50219 10.90326 11.70272 8.041499 7.489337 7.765418 Ica1 -6.77085 -1.50699 23.62132 16.63031 20.12582 13.10478 13.60514 13.35496 RP23-445K8.1 -21.306 -1.50682 65.95547 60.73287 63.34417 42.20836 41.86804 42.0382 Ccdc62 -11.8592 -1.50639 37.91747 32.63897 35.27822 22.73226 24.10584 23.41905 Trip13 -1.04168 -1.50623 3.124218 3.074565 3.099391 1.983079 2.132345 2.057712 Acyp1 -8.10933 -1.50584 23.02791 25.25376 24.14084 15.3764 16.68663 16.03151 Adss -5.2125 -1.50569 16.18676 14.85342 15.52009 8.995913 11.61928 10.3076 Pqlc1 -1.92696 -1.50561 7.317875 4.158421 5.738148 3.645646 3.976725 3.811186 Nudt4 -57.0421 -1.50557 181.3079 158.4333 169.8706 104.9864 120.6705 112.8284 Ndufa2 -9.98049 -1.50539 33.88805 25.56932 29.72869 25.36003 14.13638 19.7482 Cutc -4.25044 -1.50533 12.3863 12.93694 12.66162 7.798346 9.024011 8.411179 Idi1 -3.15927 -1.50504 8.853919 9.975592 9.414756 5.489685 7.021281 6.255483 Letm2 -20.7683 -1.50496 65.51879 58.27518 61.89698 38.22795 44.02948 41.12872 RP23-282P1.1 -1.82345 -1.50488 6.999705 3.870467 5.435086 3.663605 3.559666 3.611636 Gm20441 -1.47965 -1.50487 4.437721 4.383101 4.410411 3.885122 1.976394 2.930758 Tcp10b -20.0551 -1.50456 70.85533 48.75068 59.803 39.60796 39.88784 39.7479 Dynlrb2 -21.2517 -1.50447 75.15496 51.60261 63.37878 43.14365 41.11049 42.12707 AV039307 -2.95813 -1.50432 10.24968 7.397677 8.82368 5.423759 6.307339 5.865549 Tmbim4 -4.73134 -1.50417 11.80699 16.42446 14.11573 8.825229 9.943544 9.384386 Fbxl6 -1.63175 -1.50408 5.933378 3.804314 4.868846 3.291202 3.182983 3.237092 Tpt1 -21.6732 -1.50388 70.55977 58.81179 64.68578 39.08815 46.93696 43.01256 Gm15246 -1.08288 -1.5038 4.064924 2.399706 3.232315 1.74306 2.555808 2.149434

113

Tpst1 -5.14043 -1.5037 17.00782 13.68369 15.34575 10.29479 10.11586 10.20532 Mmadhc -15.4448 -1.50303 51.69107 40.60536 46.14822 28.6005 32.80638 30.70344 4930435E12Rik -5.85285 -1.50291 20.34391 14.6376 17.49075 9.833603 13.44221 11.63791 1700080O16Rik -5.26015 -1.50255 18.50792 12.9464 15.72716 9.576965 11.35707 10.46702 Hnrnpm -5.14384 -1.50251 18.21816 12.54194 15.38005 9.4229 11.04952 10.23621 Lrrc57 -29.1486 -1.5024 94.44754 79.88655 87.16705 56.44281 59.59408 58.01844 Nol10 -1.32449 -1.50231 3.949866 3.972717 3.961291 2.421493 2.852105 2.636799 4930474N05Rik -1.97388 -1.50223 7.700752 4.10749 5.904121 3.470025 4.390467 3.930246 Fnbp1l -2.60111 -1.50219 8.736764 6.824505 7.780634 4.865844 5.493212 5.179528 Gm3883 -1.48283 -1.50214 4.271427 4.600241 4.435834 2.394705 3.511301 2.953003 Hsbp1l1 -1.3039 -1.50214 3.873694 3.927502 3.900598 2.87491 2.318478 2.596694 Csnk2b -104.122 -1.5021 336.4746 286.5141 311.4944 217.148 197.5962 207.3721 Spz1 -217.482 -1.50182 721.2211 580.5155 650.8683 406.7966 459.9759 433.3863 Uri1 -5.46359 -1.5018 17.38355 15.31942 16.35149 9.940814 11.83498 10.8879 Ttc39d -24.0191 -1.50163 70.83062 72.97266 71.90164 42.87885 52.88625 47.88255 Ift57 -5.01732 -1.50086 16.21582 13.85368 15.03475 8.733671 11.3012 10.01744 1700021F07Rik -17.3304 -1.50061 53.66483 50.23323 51.94903 37.67245 31.56471 34.61858 Ccdc146 -1.79188 -1.5 6.851736 3.89949 5.375613 3.322477 3.844997 3.583737

114

Supplemental Table 3: Transcriptional changes of meiotic genes associated with 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Asz1 -0.77056 -1.30815 3.755357 2.7869 3.271129 2.267155 2.733985 2.50057 Aurkc 2.228795 1.328028 9.732091 3.856976 6.794534 5.437766 12.60889 9.023329 Brdt -10.6183 -1.36697 43.74474 35.36231 39.55352 27.42209 30.4484 28.93525 Bub3 -0.37877 -1.0312 11.47329 13.56698 12.52014 12.22173 12.06101 12.14137 Ccdc79 -0.62141 -1.11794 5.319584 6.461386 5.890485 4.348944 6.189205 5.269074 Ccna1 -2.91845 -1.04132 66.82936 80.26131 73.54533 59.36773 81.88604 70.62689 Ccnb3 0.321523 1.522228 0.53066 0.700692 0.615676 0.892696 0.981704 0.9372 Cdk2 -3.67107 -1.20981 22.83821 19.49799 21.1681 16.56289 18.43117 17.49703 Clasp2 0.273465 1.07518 3.439429 3.835539 3.637484 3.697645 4.124254 3.910949 Clgn -43.6139 -1.53044 140.7028 110.9699 125.8363 78.78076 85.66409 82.22242 Dmc1 0.006128 1.002295 3.358659 1.980835 2.669747 2.625192 2.726558 2.675875 Dpep3 -16.3317 -1.10031 177.625 180.6727 179.1489 160.5701 165.0643 162.8172 Exo1 0.219653 1.101477 1.963365 2.365778 2.164571 2.405949 2.3625 2.384225 Fbxo43 -1.70393 -1.29768 9.192187 5.663589 7.427888 5.06572 6.3822 5.72396 Fkbp6 -1.13787 -1.08994 11.43175 16.14763 13.78969 13.07197 12.23166 12.65182 H1foo 0.059339 2.529448 0.077595 0 0.038798 0.04991 0.146364 0.098137 H2afx 15.68773 1.295682 59.05312 47.05915 53.05613 81.85705 55.63068 68.74387 Hfm1 0.105023 1.0355 3.097994 2.81876 2.958377 2.993488 3.133312 3.0634 Hmga2 -0.01046 -1.05635 0.206421 0.185601 0.196011 0.150474 0.220637 0.185556 Hormad1 -3.62576 -1.28762 16.00814 16.45545 16.23179 11.99531 13.21675 12.60603 Hormad2 0.059591 1.012777 4.772092 4.555471 4.663782 4.560319 4.886427 4.723373 Klhdc3 -13.7175 -1.1025 139.6567 155.432 147.5443 132.2612 135.3924 133.8268 Mael -133.033 -1.47688 413.2161 410.7848 412.0005 252.8725 305.0616 278.9671 Mapk1ip1 -0.96964 -1.25557 5.097605 4.429736 4.763671 3.947315 3.640748 3.794031 Marf1 -1.22183 -1.16669 8.701134 8.402334 8.551734 6.793156 7.866648 7.329902 Mei1 -0.4585 -1.16559 3.463222 2.991681 3.227451 3.088222 2.449676 2.768949 Mei4 -0.06373 -2.47708 0.158298 0.055454 0.106876 0.043636 0.042655 0.043146 Meig1 -78.7413 -1.59858 278.7308 141.8436 210.2872 127.8252 135.2666 131.5459 Meiob -1.3676 -1.56676 4.541954 3.019278 3.780616 1.93588 2.890146 2.413013 Mnd1 -0.14736 -1.0937 2.132543 1.307356 1.71995 1.234501 1.910683 1.572592 Mns1 -9.86168 -1.3575 49.62656 25.26665 37.44661 27.97445 27.19541 27.58493 Mre11a -1.32088 -1.22914 6.852828 7.318092 7.08546 5.870283 5.658883 5.764583 Msh4 -0.38617 -1.2207 2.581537 1.690329 2.135933 1.62569 1.873827 1.749758 Msh5 0.24567 1.090525 2.698289 2.729407 2.713848 3.406035 2.513 2.959518 Nbn -2.43236 -1.32711 10.66945 9.067053 9.868253 7.099315 7.772472 7.435894 Nek2 -0.22581 -1.00425 50.49628 56.3218 53.40904 53.62383 52.74262 53.18323 Piwil1 -13.7031 -1.157 101.4386 100.5305 100.9846 86.82758 87.7354 87.28149

115

Piwil2 -1.48515 -1.0318 42.64114 53.74753 48.19434 45.5817 47.83668 46.70919 Piwil4 -0.07561 -2.01394 0.173059 0.127312 0.150186 0.100181 0.048965 0.074573 Pld6 -1.40111 -1.05178 25.51248 31.41092 28.4617 26.50816 27.61301 27.06058 Ppp2ca 0.095197 1.046022 1.741909 2.395097 2.068503 2.144783 2.182617 2.1637 Prdm9 -0.476 -1.59885 1.427735 1.113984 1.270859 0.834845 0.75487 0.794858 Psmc3ip -8.60192 -1.44224 31.7817 24.32329 28.0525 20.04657 18.85459 19.45058 Rad21l -0.14969 -1.13112 1.0673 1.515241 1.29127 1.011677 1.271485 1.141581 Rad50 0.615488 1.154991 4.455885 3.486348 3.971116 4.839959 4.33325 4.586604 Rbbp8 -1.68985 -1.29495 7.687689 7.150666 7.419178 5.208294 6.250358 5.729326 Rbm7 -5.17167 -1.25936 26.90029 23.32285 25.11157 17.73586 22.14394 19.9399 Rec114 -0.44884 -1.96599 1.021684 0.805299 0.913491 0.516336 0.412959 0.464647 Rec8 -4.00243 -1.13712 32.82751 33.55538 33.19144 28.79304 29.58499 29.18901 Rnf212 -0.92052 -1.3368 4.530174 2.777224 3.653699 3.330105 2.136249 2.733177 Rsph1 -151.153 -1.27523 702.1436 698.5351 700.3394 574.649 523.724 549.1865 Sgol2 -0.53885 -1.08219 6.509676 7.680763 7.095219 5.958993 7.153744 6.556368 Sirt2 -5.61717 -1.13249 47.51023 48.5169 48.01357 44.78841 40.00438 42.3964 Slc26a8 -7.11064 -1.18149 43.77511 48.80577 46.29044 37.22163 41.13797 39.1798 Smc1a 0.329379 1.484322 0.904621 0.455546 0.680084 1.028644 0.990283 1.009463 Smc1b -1.56248 -1.12077 17.28601 11.71492 14.50047 11.44507 14.43091 12.93799 Smc3 0.300827 1.063067 5.415606 4.12427 4.769938 5.192583 4.948947 5.070765 Spata22 -0.35466 -1.16437 2.539885 2.484697 2.512291 1.894365 2.4209 2.157632 Spin1 -1.00053 -1.09536 10.71899 12.26638 11.49269 9.665865 11.31845 10.49216 Spo11 -0.70128 -1.06506 12.38284 10.57828 11.48056 11.06808 10.49048 10.77928 Stag2 -0.60498 -1.22301 3.63847 2.997105 3.317788 2.535919 2.889697 2.712808 Stag3 -1.63862 -1.01783 87.76285 99.275 93.51892 90.04352 93.71709 91.88031 Stra8 -2.13519 -1.91588 4.496792 4.436164 4.466478 2.127721 2.53486 2.33129 Syce1 -57.3688 -1.48563 199.7703 151.2303 175.5003 111.5198 124.7433 118.1315 Syce2 -6.48345 -1.37027 25.80778 22.17908 23.99343 16.17334 18.84663 17.50999 Syce3 -1.91751 -1.11097 23.92999 14.46366 19.19683 17.23465 17.32398 17.27931 Sycp1 -8.60927 -1.928 19.54947 16.22354 17.88651 7.098151 11.45631 9.277232 Sycp2 -8.06342 -1.42924 30.22068 23.47684 26.84876 15.9624 21.60829 18.78535 Sycp3 -9.92889 -1.73699 30.89202 15.91003 23.40102 10.8123 16.13197 13.47213 Tdrd1 -0.86025 -1.02448 34.63673 37.36689 36.00181 32.94986 37.33326 35.14156 Tdrd12 -0.62063 -1.05204 13.21316 11.88227 12.54771 10.87678 12.97739 11.92708 Tex11 -0.33336 -1.21589 1.800069 1.954829 1.877449 1.439002 1.649176 1.544089 Tex19.1 0.780623 1.133418 5.476034 6.225864 5.850949 6.596169 6.666976 6.631572 Tex19.2 -0.50986 -1.0402 11.97924 14.40852 13.19388 13.1251 12.24294 12.68402 Trip13 -1.04168 -1.50623 3.124218 3.074565 3.099391 1.983079 2.132345 2.057712 Utp14b -0.02946 -1.26898 0.218448 0.05952 0.138984 0.070254 0.148794 0.109524 Wee2 0.053441 1.350633 0.153884 0.150942 0.152413 0.23755 0.174157 0.205854 Zfp318 -1.52264 -1.05063 30.64475 32.54778 31.59627 29.80354 30.3437 30.07362

116

Supplemental Table 4: Transcriptional changes of tight junction genes associated with 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Amot 0.112683 1.067836 1.486283 1.835931 1.661107 1.612338 1.935243 1.77379 Amotl1 -2.52921 -1.32125 11.81309 8.991295 10.40219 8.24675 7.499205 7.872977 Arhgap17 -0.32268 -1.14737 2.600953 2.423662 2.512308 2.392322 1.986939 2.189631 Arhgef2 -2.46821 -1.08898 25.93796 34.4738 30.20588 28.82841 26.64694 27.73767 Ash1l 0.106337 1.019681 5.580411 5.225545 5.402978 5.070031 5.948599 5.509315 Bves -0.00471 -1.04618 0 0.21331 0.106655 0.149202 0.054693 0.101947 Ccdc85c -1.48237 -1.21645 7.650551 9.011038 8.330794 7.788183 5.908665 6.848424 Cgn -1.08313 -1.0937 12.65726 12.62826 12.64276 12.60097 10.51829 11.55963 Cgnl1 -0.64667 -1.18589 4.740396 3.510628 4.125512 3.629831 3.327861 3.478846 Cldn1 -0.06141 -2.34888 0.143505 0.070381 0.106943 0.036921 0.054137 0.045529 Cldn10 -0.03141 -1.22021 0.215806 0.1323 0.174053 0.115673 0.169609 0.142641 Cldn11 -1.50591 -1.0668 23.50019 24.59999 24.05009 19.73141 25.35695 22.54418 Cldn12 0.283375 1.164923 1.408277 2.028174 1.718225 1.768494 2.234706 2.0016 Cldn13 -0.26972 -1.22615 1.746807 1.177968 1.462388 1.067381 1.317959 1.19267 Cldn14 -0.10487 -2.72074 0.331645 0 0.165822 0.121895 0 0.060948 Cldn15 -0.07595 -2.07656 0.131627 0.161388 0.146508 0.141106 0 0.070553 Cldn16 0 1 0 0 0 0 0 0 Cldn17 0 1 0 0 0 0 0 0 Cldn18 0.020214 1.86836 0.020914 0.025642 0.023278 0.06726 0.019724 0.043492 Cldn19 0.00703 (∞) 0 0 0 0.014061 0 0.00703 Cldn2 -0.01476 -1.33204 0.084083 0.034365 0.059224 0.036055 0.052867 0.044461 Cldn22 -0.16174 -1.57484 0.656283 0.229905 0.443094 0.120607 0.44211 0.281359 Cldn23 0.067454 1.102036 0.704857 0.617304 0.66108 0.744821 0.712248 0.728534 Cldn3 -2.95285 -1.2841 14.80415 11.88915 13.34665 11.71223 9.075363 10.3938 Cldn4 0.039395 2.529448 0.051516 0 0.025758 0.033135 0.097171 0.065153 Cldn5 -0.44347 -1.1119 5.338447 3.474756 4.406602 4.19678 3.729479 3.963129 Cldn6 -0.02155 -1.27542 0.123761 0.075872 0.099816 0.039802 0.116722 0.078262 Cldn7 0.033382 1.147644 0.334879 0.117313 0.226096 0.338481 0.180475 0.259478 Cldn9 -0.21413 -1.86407 0.844249 0.079626 0.461938 0.250628 0.244993 0.247811 Clmp -0.02023 -1.05456 0.284184 0.497769 0.390977 0.339466 0.40203 0.370748 Crb3 -0.02055 -1.0987 0.238343 0.219174 0.228759 0.19163 0.224786 0.208208 Cxadr 0.036217 1.271898 0.103399 0.163 0.133199 0.199521 0.139311 0.169416 Ddx58 -0.11825 -1.21236 0.766687 0.58347 0.675079 0.552656 0.561009 0.556832 Ect2 -1.17824 -1.14218 8.563215 10.36757 9.465392 7.247875 9.326423 8.287149 Epcam -0.37182 -1.05588 8.451249 5.601123 7.026186 6.199871 7.108863 6.654367 Esam -0.16408 -1.3148 0.812628 0.557963 0.685296 0.459966 0.582468 0.521217 F11r 1.217712 1.173793 6.719073 7.29429 7.006682 7.405512 9.043275 8.224394

117

Igsf5 -0.12372 -1.32503 0.471688 0.537027 0.504358 0.411748 0.349532 0.38064 Inadl -0.41606 -1.357 1.573855 1.589168 1.581511 1.108508 1.222393 1.16545 Jam2 -0.23686 -1.10618 2.269495 2.665875 2.467685 2.051825 2.409829 2.230827 Lin7a -4.39764 -1.40689 16.20529 14.20607 15.20568 11.27939 10.33669 10.80804 Lin7b -0.30162 -1.80317 0.573294 0.781018 0.677156 0.450691 0.300381 0.375536 Lin7c -1.37424 -1.30158 5.998433 5.863525 5.930979 4.136666 4.976819 4.556742 Magi1 0.254613 1.23388 1.198029 0.979269 1.088649 1.214249 1.472275 1.343262 Magi3 0.219165 1.106177 2.036385 2.091924 2.064155 2.256781 2.309858 2.28332 Marveld2 -0.05371 -1.13903 0.440885 0.439213 0.440049 0.460819 0.311856 0.386337 Marveld3 -0.17489 -2.14084 0.48242 0.173969 0.328194 0.146022 0.160581 0.153301 Micall2 -0.241 -1.27839 1.056193 1.157232 1.106712 0.968436 0.762983 0.86571 Mpdz -0.01425 -1.04203 0.329698 0.376681 0.353189 0.395211 0.282677 0.338944 Mpp5 -1.64552 -1.17855 11.19253 10.53076 10.86164 8.243096 10.18915 9.216124 Mpp7 -0.21897 -1.12809 1.835745 2.021133 1.928439 1.722953 1.695995 1.709474 Mtdh -1.10417 -1.31523 4.830644 4.383101 4.606873 3.161617 3.843795 3.502706 Nphp1 -110.372 -1.72202 318.8768 207.5973 263.2371 150.2974 155.4328 152.8651 Nphp4 -0.63263 -1.15529 4.85697 4.556149 4.706559 4.076127 4.071739 4.073933 Ocln -0.4208 -1.37827 1.280233 1.786204 1.533218 0.809258 1.415589 1.112423 Pard3 -0.04253 -1.09867 0.513951 0.433232 0.473591 0.347106 0.515012 0.431059 Pard3b 0.134271 1.128943 0.970678 1.111963 1.04132 0.947914 1.403269 1.175591 Pard6a -5.13484 -1.24853 22.02723 29.56453 25.79588 19.9946 21.32749 20.66105 Pard6b -0.13325 -1.06678 1.836974 2.420397 2.128685 1.922232 2.068641 1.995436 Pard6g -0.63044 -1.41412 2.342405 1.963155 2.15278 1.506652 1.53803 1.522341 Plxdc1 -3.59287 -1.25991 17.84448 16.98822 17.41635 13.90711 13.73985 13.82348 Pof1b 0.008436 1.573788 0 0.029404 0.014702 0.046275 0 0.023138 Rab13 -0.46579 -1.22123 3.273861 1.868624 2.571243 1.81532 2.395586 2.105453 Rpgrip1l -0.57004 -1.18032 4.018941 3.44354 3.73124 3.13851 3.183889 3.161199 Shroom2 0.094175 1.217249 0.38946 0.477517 0.433488 0.572579 0.482747 0.527663 Sympk 1.366726 1.028038 46.37439 51.11491 48.74465 53.85289 46.36986 50.11138 Synpo -0.02189 -1.01481 1.681428 1.318464 1.499946 1.351883 1.604228 1.478056 Tbcd 0.334867 1.100874 2.969809 3.669511 3.31966 3.346558 3.962495 3.654526 Tgfbr1 -1.34571 -1.30367 5.97605 5.578492 5.777271 4.128229 4.73489 4.431559 Tjap1 -1.47722 -1.14876 10.48636 12.32895 11.40765 9.632279 10.22859 9.930437 Tjp1 -1.99793 -1.16755 14.72588 13.1185 13.92219 11.19024 12.65827 11.92425 Tjp2 -2.60063 -1.47266 9.012702 7.192765 8.102733 5.509796 5.494409 5.502102 Tjp3 0.050984 1.444003 0.154088 0.075571 0.114829 0.09911 0.232517 0.165814 Traf4 -0.03235 -1.02219 1.556977 1.42308 1.490028 1.420251 1.495115 1.457683 Ubn1 -0.23425 -1.01477 15.49112 16.69248 16.0918 15.08994 16.62516 15.85755 Vasp -6.57359 -1.23086 38.22483 31.86989 35.04736 27.24184 29.70571 28.47377 Wnk4 0.315298 1.105532 3.05548 2.91993 2.987705 3.236977 3.369029 3.303003

118

Supplemental Table 5: Transcriptional changes of vitamin A metabolism and signaling genes associated with 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Adh4 -2.43072 -1.57525 3.856227 9.456241 6.656234 2.935085 5.515943 4.225514 Aldh1a1 16.73987 1.252186 63.18727 69.5707 66.37898 80.05876 86.17895 83.11886 Aldh1a2 -2.39123 -1.0483 50.30071 53.49765 51.89918 48.16002 50.85589 49.50795 Aldh1a3 -0.45567 -1.83015 0.969279 1.039879 1.004579 0.545517 0.552297 0.548907 Aldh8a1 -0.22636 -1.66485 0.743159 0.390509 0.566834 0.300461 0.380482 0.340471 Bcmo1 -3.706 -2.00029 9.564746 5.257083 7.410914 3.677127 3.732706 3.704916 Crabp1 -1.0664 -1.31293 4.531788 4.416646 4.474217 4.185468 2.630166 3.407817 Crabp2 -0.10089 -1.75014 0.21148 0.259296 0.235388 0.136025 0.132967 0.134496 Cyp26a1 -0.55876 (-∞) 0.473057 0.644461 0.558759 0 0 0 Cyp26b1 0.786747 1.085757 8.686915 9.661331 9.174123 9.209466 10.71227 9.96087 Cyp26c1 0.05031 (∞) 0 0 0 0 0.10062 0.05031 Dhrs3 0.258768 1.173437 1.30539 1.678615 1.492003 2.15029 1.351251 1.75077 Dhrs4 -3.62301 -1.44325 13.81121 9.782221 11.79672 8.064124 8.28329 8.173707 Lrat -0.12883 -2.65899 0.156743 0.256243 0.206493 0.089616 0.065701 0.077658 Rara 0.251808 1.067832 3.864325 3.560082 3.712203 3.941196 3.986826 3.964011 Rarb 0.005028 1.018384 0.245727 0.301287 0.273507 0.296351 0.260719 0.278535 Rarg -0.01084 -1.01282 0.9072 0.806431 0.856816 0.729397 0.962549 0.845973 Rbp1 -0.56522 -1.43928 1.721876 1.981936 1.851906 1.446561 1.126813 1.286687 Rbp2 0.048603 1.818395 0 0.118776 0.059388 0.124619 0.091363 0.107991 Rbp3 -0.02654 -1.26247 0.211961 0.043314 0.127638 0.102251 0.099952 0.101102 Rbp4 -1.07867 -1.76202 2.874083 2.114348 2.494216 1.340258 1.490833 1.415546 Rbp7 -0.39535 -4.8403 0.519176 0.477422 0.498299 0.083485 0.122411 0.102948 Rdh10 -0.45719 -1.23801 2.531421 2.224831 2.378126 1.729093 2.112774 1.920933 Rdh11 -10.7611 -1.13926 82.80749 93.26235 88.03492 71.94761 82.6001 77.27386 Retsat 0.919402 1.151966 6.349818 5.750296 6.050057 7.304199 6.634719 6.969459 Rxra -0.32978 -1.04145 8.30915 8.263246 8.286198 7.802761 8.110081 7.956421 Rxrb 0.064382 1.01158 4.939335 6.179714 5.559525 5.900175 5.347638 5.623906 Rxrg -0.20679 -1.62564 0.527426 0.547189 0.537307 0.469723 0.191318 0.33052 Stra6 0.938257 2.04391 0.719445 1.078137 0.898791 1.78931 1.884787 1.837049 Stra8 -2.13519 -1.91588 4.496792 4.436164 4.466478 2.127721 2.53486 2.33129

119

Supplemental Table 6: Transcriptional changes of steroidogenic genes associated with 8 days of WIN 18,446 treatment

Feature ID Difference Fold Veh - 1 Veh - 2 Veh - WIN - 1 WIN - 2 WIN - (WIN- Change Means Means Veh)

Cyp11a1 -2.9131 -1.20254 16.37264 18.21927 17.29596 13.5256 15.24012 14.38286 Cyp11b2 -0.0539 -1.12673 0.114698 0.843788 0.479243 0.147549 0.703131 0.42534 Cyp17a1 -6.57598 -1.10201 71.55057 70.53071 71.04064 58.97329 69.95603 64.46466 Cyp21a1 0.151575 1.28807 0.304834 0.747514 0.526174 0.616225 0.739273 0.677749 Fdx1 -2.91541 -1.37665 9.633102 11.67844 10.65577 5.987226 9.493496 7.740361 Hsd3b1 -0.31003 -1.02353 12.55959 14.41376 13.48667 11.66524 14.68805 13.17664 Hsd3b2 0.009616 1.017997 0.424964 0.643648 0.534306 0.498444 0.5894 0.543922 Hsd3b3 -0.00149 -1.06031 0.052503 0 0.026251 0 0.049516 0.024758 Hsd3b4 0 1 0 0 0 0 0 0 Hsd3b5 -0.194 (-∞) 0.1743 0.213709 0.194004 0 0 0 Hsd3b6 8.821843 1.167202 45.66519 59.85804 52.76161 60.54064 62.62628 61.58346 Hsd3b7 -0.09267 -1.07172 1.711198 1.058334 1.384766 1.655853 0.92833 1.292091 Pbx1 0.133826 1.180008 0.715833 0.771049 0.743441 0.770251 0.984283 0.877267 Star -2.29693 -1.10606 20.93229 26.97704 23.95466 18.02343 25.29203 21.65773 Stard3 -1.03057 -1.30354 4.611138 4.240288 4.425713 3.083881 3.706413 3.395147

120

CHAPTER 3

INCREASED TESTICULAR RA HAS ABNORMAL EFFECTS ON SPERMATOGENESIS

Increased testicular RA has abnormal effects on spermatogenesis

Travis Kent2, Alyssa Marre2, Cathryn A. Hogarth2, Faith Stevisond3, Debra Mitchell2, Nina

Isoherranen3, and Michael D. Griswold2

2School of Molecular Biosciences and The Center for Reproductive Biology, Washington State

University, Pullman, Washington, USA.

3Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.

Summary sentence: RA degradation likely takes place predominantly in PTMs and increased RA

levels causes spermatogenic abnormalities.

Key words: CYP26, testis, meiosis, talarozole, Sertoli cell, retinoic acid.

122

Abstract

Retinoic acid (RA) is vital for the proper production of spermatozoa in the mammalian testis. While the importance of this molecule has been well documented, the manner in which it is regulated is not well understood. There has been comparatively little work done on RA degradation, which could be a key factor in cell specific RA availability. Additionally, inhibition of RA degradation has been shown to increase RA levels in many organs, but the testis remains uninvestigated. The data presented here shows isozyme specific testicular localization via immunohistochemistry of the CYP26 enzymes, which are responsible for RA degradation, to the peritubular myoid cells. Additionally, treatment with a potent CYP26 inhibitor resulted in misregulation of spermatogonial differentiation in adults, and drove synchronized spermatogenesis in neonates. Finally, treatment with exogenous RA resulted in a reduction in recombination rates and decreased Sertoli cell number. Taken together, these data show that an increase in testicular RA levels, and specifically an inhibition of RA degradation, has abnormal effects on proper sperm production.

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Introduction

The generation of spermatozoa in the mammalian testis is an extremely complex process, requiring the precise regulation of many specific molecules, namely retinoic acid (RA). Both low and high levels of RA, the active metabolite of vitamin A, have been shown to have adverse effects on spermatogenesis. For example, the absence of testicular RA prevents spermatogonial differentiation, resulting in testes devoid of advanced germ cells, causing sterility [1-7].

Conversely, exogenous RA embryonically causes premature meiotic initiation [8]. These data highlight the importance of proper regulation of testicular RA levels.

Ingested through the diet, vitamin A, in the form of retinol, is transported throughout the body via the serum. Once it reaches its target tissue, ROL is converted to RA by way of a two- step enzymatic process [9]. RA then either signals through the retinoic acid/retinoid X receptor heterodimers, or is degraded by the CYP26 family of enzymes, namely CYP26A1 and

CYP26B1, to inert metabolites [9]. The cell-specific localization patterns of these enzymes are important for precise RA degradation throughout the testis. In the embryo, RA is thought to play a role in tissue development and organogenesis, based largely on mRNA localization data [8, 10-

12]. While the Cyp26 transcripts have been localized to the peritubular myoid cells (PTM) [13], complete localization of the enzymes in the testis has not been completed.

Why is elucidating these localization patterns important? Hogarth et al. 2015 recently reported a pulse of RA present across the spermatogenic cycle, but the regulation of this pulse is unknown [14]. Investigating the presence of RA degradation enzymes in the context of the spermatogenic cycle could clarify how this pulse is regulated. Additionally, a potent CYP26 inhibitor, talarozole (TAL), is being used for the clinical treatment of dermatological diseases such as acne and psoriasis [15]. The testicular CYP26 localization would provide context about

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where this inhibitor could be having an effect within the testis. It is also notable that TAL is being used clinically despite the fact that the effects of this potent inhibitor on testicular function have not been investigated.

TAL treatment has been previously shown to raise endogenous RA levels within many organs, including the liver and kidney [16], but testicular RA levels were not assessed. While not as well studied as the effects of absent RA, there are data describing the effects of excess RA on spermatogenesis. For example, Snyder et al. 2011 reported that adult vitamin A-sufficient animals treated with RA exhibited a significant increase in apoptotic spermatogonia [17]. When these animals were analyzed one month later, approximately a quarter of the seminiferous tubules exhibited a missing layer of round spermatids at Stages VII and VIII [17]. These data indicate that high levels of exogenous RA ablate a cohort of spermatogonia at specific stages of the cycle but subsequent differentiation appears to proceed normally. Another study looked at the dosage dependent effects of RA on cultured TM-3 cells, a Leydig cell line [18]. They found that at higher-than-physiological doses, Leydig cell apoptosis increased [18]. A separate study of immature rat Sertoli cells cultured with RA resulted in premature maturation [19], however it is not currently known if increased testicular RA causes premature Sertoli cell maturation in vivo. Finally, it was also shown that excess RA in the lizard Podarcis sicula resulted in a drastic reduction of germ cells, specifically spermatocytes and spermatogonia, likely due to apoptosis

[20]. These data, when taken together, show that increases in RA have an adverse effect on spermatogenesis.

While there has been no work investigating the effects of global CYP26 inhibition in the postnatal testis, there has been some data generated in the embryonic gonad investigating the effect of excess RA on gametogenesis. Specifically, CYP26 activity in the embryonic testis is

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important for RA degradation throughout the embryo, proper gonadal formation, and prevention of premature entry into meiosis [21-23]. Cyp26b1 null male embryos are incapable of degrading

RA efficiently within their testes and, therefore, the germ cells enter meiosis [8, 21].

Additionally, Cyp26a1 null animals fail to develop a functional urogenital system, among other severe morphological defects [22]. There has been some work looking at cell-specific Cyp26 knockout animals. Spermatogenesis was only disrupted in animals null for Cyp26b1 in either

Sertoli or germ cells [24, 25], indicating that this isozyme is important within the seminiferous tubule. Thus far however, no postnatal global Cyp26 ablations have been published.

Thus far, only a cursory investigation into the effects of exogenous RA on spermatogenesis has been conducted. There are no reports on what effects excess RA has on

Sertoli cells or meiosis and, while potent inhibitors of RA degradation are being used clinically, the consequences of these compounds on fertility have not been investigated. The goal of this study was to determine the testicular localization of the CYP26 proteins, investigate the effects of TAL treatment on testicular RA levels, and elucidate the effects of increased testicular RA levels on spermatogonial differentiation, Sertoli cell maturation, and meiotic recombination. The results of this study show that CYP26 enzymes are present in every stage of spermatogenesis and that their inhibition causes precocious spermatogonial differentiation. Additionally, excess RA causes a decrease in Sertoli cell and meiotic recombination.

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Materials and Methods

Animal care and handling

Following the approval of the Washington State University Animal Care and Use

Committee, C57BL/6-129 mice were used for all experiments. Colonies were housed in a temperature- and humidity-controlled environment and food and water freely provided. Mice were treated as described below and euthanized via two separate methods: CO¬2 asphyxiation followed by either decapitation (0-10 days post-partum [dpp]) or cervical dislocation (> 10 dpp).

Tissue was subsequently dissected from the mouse.

WIN 18,446, RA, and TAL treatments

To assess the effects of excess RA on meiotic initiation, spermatogenesis was synchronized as previously described [7]. Briefly, 2 dpp male mice were treated with 100 μg/g body weight WIN 18,446 for 7 days. On the following day (8th day of treatment, 9 dpp), the animals were treated with 200 μg RA (Sigma Aldrich). Five days after RA treatment, these animals were either treated with 100 μg RA/day or DMSO as the vehicle control for seven days.

The following day these animals were euthanized and their testes were extracted for further analysis.

To determine the consequences of excess RA on Sertoli cell maturation, animals were treated with 50 μg RA/day, or DMSO as the vehicle control, from 5-8dpp. At 9 dpp, animals were injected with 20 μL EdU (Molecular Probes, 10 mM) and then euthanized after 4 h of EdU incorporation.

Assessment of CYP26 inhibition on spermatogonial differentiation was investigated by mimicking previously published experiments utilizing exogenous RA [17]. Briefly, adult mice were treated via IP injections with 5 mg/kg/dose TAL, 100 μg RA, or DMSO as the vehicle

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control twice per day for three days. Twelve hours following the last dose, the animals were euthanized and the testes collected for further analysis. To determine if TAL was sufficient to synchronize spermatogenesis, neonatal animals were treated orally with 5 mg/kg TAL or PEG

300 as vehicle control twice daily from 2 dpp to 4 dpp. These animals were then sacrificed 47 days later. There testes were dissected for further analysis.

Immunoblotting

Western blots were performed using rabbit polyclonal antibodies specific to CYP26A1 and CYP26B1 [26, 27]. Briefly, 50 μg of adult mouse testis protein was loaded onto and separated via SDS-PAGE (Bio-Rad Laboratories, #456-1084) and transferred to a nitrocellulose membrane. The membrane was then washed briefly with Tris-buffered saline (TBS) (50 mM

Tris Base, 0.9% NaCl, pH 7.5) and blocked with 5% skim milk in TBS-T (TBS + 0.1% Tween-

20) for an hour at room temperature. Antibodies were diluted (1:1000) in 5% skim milk/TBS-T and applied to the membranes for approximately 20 h at 4°C. The membranes were then washed three times with TBS-T for 5 min each at room temperature. Horseradish peroxidase goat anti- rabbit secondary antibody (Cell Signaling Technology, #7074, 1:1000 dilution) was applied to membranes for one hour at room temperature. Membranes were again washed three times with

TBS-T for 5 min each before they were imaged via chemilumenescence. The membranes were exposed for 15 s and imaged on Fujifilm LAS-4000.

Immunohistochemistry

Immunohistochemistry was performed as previously described [28] using mouse testis tissue (n

= 3) fixed in Bouin’s fixative for 1-6 h depending on the age of the animal, embedded in paraffin, and sectioned onto charged glass slides using the same antibodies specific for

CYP26A1 and CYP26B1, as well as STRA8 [14] and SOX9 [29] (Merk Millipore, AB5535).

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Antigen retrieval was achieved using citrate buffer (10 mM, pH 6) at a rolling boil for 5 min.

Antigen retrieval was not used when probing for STRA8. Sections were incubated in primary antibody at a concentration of 1:500 (SOX9) or 1:1000 (CYP26A1, CYP26B1, and STRA8) in

5% normal goat serum/ 0.1% bovine serum albumin in phosphate buffered saline (PBS) (137mM

NaCl/2.7mM KCl/10.1mM Na 2HPO4/1.8mM KH2PO4) at 4°C overnight (~16 h). Control sections were incubated without primary antibody. Biotinylated or fluorophore conjugated goat- anti-rabbit secondary antibody (Intvitrogen, 956143b, Alexa Fluora Cat #) was applied for 1 h at room temperature, following the manufacturer’s instructions. Strepdavidin conjugated horseradish peroxidase (HRP) (Invitrogen, 956143b) was also applied for 1 h at room temperature. Binding was determined by a brown precipitate formed by HRP activity in the presence of 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Invitrogen, 002020). If DAB stained, sections were counterstained with a 1:3 dilution Harris Heamotoxilin (Sigma-Aldrich,

HHS32-1L) and dehydrated. Slides were then mounted under glass coverslips using DPX mounting media (VWR International, 360294H) or DAPI (Abcam, ab104139). Cell types were determined using nuclear morphology, location within the testis, and cellular markers such as

STRA8 and SOX9 [30]. EdU incorporation was visualized following the manufacturer’s instruction included in the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Molecular Probes,

C10337)

Generation of subcellular fractions from mouse tissue

Mouse testis S10 fractions containing microsomes and cytosol were separately generated from 48 samples containing pooled testes from neonatal mice (24-50 mg) and 40 samples containing individual testes from adult mice (43-89 mg), using a previously described method

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[8]. The total protein concentration in each S10 fraction was measured using a BCA assay

(Thermo Scientific, PI-23227).

Mass spectrometric quantification of RA

The concentrations of RA in incubations and tissue samples were measured using an AB

Sciex 5500 qTrap Q-LIT mass spectrometer equipped with an Agilent 1290 UHPLC as previously described [14, 31]. For quantification, RA peak areas were normalized to the atRA- d5 internal standard peak area. All data analysis was performed using Analyst (version 1.5.1)

(AB Sciex). A signal:noise ratio of 9 was set as the minimum threshold for quantitation.

Adult mouse testis staging and analysis

The stage distribution of both TAL and control treated animals was determined using previously established guidelines [30, 32, 33]. Stage distribution was determined by analyzing at least 200 tubules from at least two histological cross sections separated by 50 μm. The midpoint of synchrony, window width, and synchrony factor were determined as previously described [14,

32, 33]. The mean synchrony factors for the TAL and control treated animals were determined and an unpaired two-tailed student t-tests were conducted to determine significance.

Meiotic Preparations and Immunostaining

Synchronized testes treated with either TAL (n=4) or vehicle (n=3) for 7 days were placed in PBS. Testes from untreated 21 dpp animals (n=3) were used as controls. Meiotic preparations were made as described in [34], with one minor modification: instead of dipping the slide in 1% paraformaldehyde, a thin layer was spread across an uncharged glass slide using a glass Pasteur pipette. Slides were allowed to incubate in a humid chamber overnight, dried, washed in 0.4% Photo-flo 200 solution (Kodak professional), air dried, and immunostained.

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Cruz Biotechnology, sc-74569, at 0.5 μg/mL) and MLH1 (Calbiochem, PC56, at 1.3 μg/mL);

MLH1 primary antibody in ADB was applied to the slide, covered with a glass coverslip, sealed with rubber cement, and incubated at 37°C for approximately 16 h. Following brief ADB wash,

(Jackson Immunoresearch Laboratories, Inc., 715-165-150, at 625 μg/mL) was applied with parafilm coverslip for 45 min at 37° C. Finally, slides were washed in PBS, and mounted with

20 μL of Prolong Gold anti-fade reagent with DAPI (Life Technologies, P36931) using glass coverslips. Slides were dried in the dark and stored at 4°C.

Recombination Analysis

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unpaired two-tailed student t-test was conducted (MS Excel 2010) to compare the recombination rate in WIN 18,446 and vehicle treated animals. The number of MLH1 foci per synaptonemal complex (SC) per cell was also assessed. A chi-squared analysis was used to determine if the distribution of MLH1 foci along the SCs in the treated animals differed significantly from those in the control treated animals.

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Results

CYP26 protein localizes predominantly to the PTM cells

While some work had been conducted regarding the testicular localization of the CYP26 family of enzymes, a more comprehensive analysis was needed. Immunoblots specific to

CYP26A1 and CYP26B1 were performed on adult mouse testis tissue to ensure that the antibodies were detecting bands of the proper size (Figure 1). The CYP26A1 antibody was able to detect a band of approximately 54 kDa, while CYP26B1 antibody detected a band of approximately 64 kDa. The expected molecular weights of CYP26A1 and CYP26B1 were 56 and 58 kDa, respectively. No other bands were detected.

Immunohistochemistry was performed on testis tissue from 0-30 dpp mice (n=3) so the postnatal testicular enzyme localization could be determined (Figure 2). CYP26A1 localized to

Sertoli, Leydig, and PTM cells at every time point analyzed (Figure 2 A, C, E, G, I, and K).

CYP26B1 was also localized to the Leydig and PTM cells, but only beginning at 15 dpp (Figure

2 B, D, F, H, J, and L).

Immunohistochemistry was also performed on adult mouse testis cross sections to determine if these enzymes were present in a stage-specific manner (Figure 3). Similar to the neonatal animals, CYP26A1 and CYP26B1 were both localized to Leydig and PTM cells.

CYP26A1 was also localized to Sertoli cells. There was no observed stage-specificity associated with the localization patterns for either CYP26A1 or CYP26B1.

TAL is metabolized quickly and in a variable manner

TAL treatments in mice are known to increase RA levels in various organs [16], but the metabolism of this molecule has not been extensively investigated in the murine model. In order to rectify this, adult male mice were treated orally with a single dose of TAL and euthanized at

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various time points within 12 hours of dosing (n=3 for each time point). Serum TAL levels were assayed using tandem HPLC-MS/MS (Figure 4). Serum TAL spiked very quickly; the highest

TAL levels were detected 30 min after the treatment. Serum TAL levels were also cleared quickly. By one hour post treatment, serum TAL levels were less than half of the maximum levels measured. There was also high variability in serum TAL measurements, indicating that

TAL treatment could have variable effects in vivo. This high measured variability also prevented statistical significance from being achieved.

TAL causes precocious spermatogonial differentiation

Because serum TAL levels were so mercurial, more functional assays were conducted to determine if TAL was having any effect on spermatogenesis. Adult mice were treated twice per day for three days with TAL. The testes of these animals were collected at various time points within 9 hours of the last treatment (n=3 for each time point) and RA measurements and Real

Time RT-PCR was conducted. Peak RA levels were observed 6 h after TAL treatment (Figure

5A), but high variability again prevented statistical significance from being achieved at any time point. Real Time RT-PCR was conducted using primers specific to Cyp26a1, a known RA responsive gene. Peak Cyp26a1 levels were detected, again, at 6 h post TAL treatment (Figure

5B). Statistical significance was not achieved in this assay as well due to high variability.

The data, thus far, showed a variable response to TAL treatment. To determine if TAL is having a consequential effect on spermatogenesis, spermatogonial differentiation was analyzed

(Figure 5C-F). Adult animals were treated with TAL, RA, or vehicle twice daily for 3 days.

Testes were collected 12 h following the final dose and immunohistochemistry specific to

STRA8, a known marker of differentiating spermatogonia, was performed. Histological analysis showed that STRA8 positive spermatogonia were present in every tubule in animals treated with

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either RA or TAL (Figure 5C-E). The number or STRA8-positive spermatogonia were quantified for each spermatogenic stage (Figure 5F). Based on previously published STRA8 localization [14, 35], the stages were sorted into three bins: Stages XI-IV, Stages V-VII, and VII-

X. These bins correspond to when spermatogonia are the only STRA8-positive cells, no cells are

STRA8 positive, and when both spermatogonia and spermatocytes are STRA8-positive, respectively. Quantification of these data showed a significant increase in STRA8-positive spermatogonia in both Stages XI-IV and Stages V-VII for animals treated with either RA or TAL when compared to control (Figure 5F). Additionally, animals treated with TAL showed a significant increase in the number of STRA8-positive spermatogonia in Stages VII-X (Figure

5F).

Neonatal TAL treatment synchronizes spermatogenesis

To determine if inhibition of RA degradation could recapitulate the phenotypes seen with exogenous RA treatment in the first wave of spermatogonial differentiation, 1 dpp mice were treated with TAL for 3 days. These animals were allowed to recover for 42 days before being euthanized. Their testes were collected and analyzed for histology. STRA8 staining was used to aid in determining spermatogenic stages (Figure 6A-B). The stage distribution and synchrony factor were quantified for vehicle (n = 3) and TAL (n = 5) treated animals (Fig 6C-D). The animals treated with TAL showed a vastly altered stage distribution. The vehicle treated animals had a calculated synchrony factor of 1.18, indicating that no synchrony had taken place. The

TAL treated animals, however, had an average synchrony factor of 4.76, indicating that spermatogenesis had been synchronized.

Exogenous RA treatment lowers meiotic recombination rates

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The previous chapter showed that decreases in testicular RA levels resulted a modest increase in recombination rate. Additionally, when the lizard, Podarcis sicula, was treated with

RA, the authors reported a reduction in the numbers of spermatocytes [20]. Taken together, these data suggest that proper testicular RA levels are important for meiotic progression. To test if increased testicular RA has an effect on meiotic recombination, animals with synchronized spermatogenesis were treated daily with either exogenous RA or vehicle 5-11 days post synchrony. Animals with synchronized spermatogenesis were used to enrich for meiotic cells in mid-pachytene stage. Additionally, synchronizing spermatogenesis was important ensure that all cells being analyzed initiated meiosis in a high testicular RA environment. The recombination analysis revealed that vehicle treated animals had approximately 23.15 MLH1 foci/cell while RA treated animals had 22.46 MLH1 foci/cell, a difference of 0.51 foci/cell (p=0.050) (Table 1).

Exogenous RA treatment decreases Sertoli cell proliferation

Increased RA levels in vitro have been shown to prematurely decrease Sertoli cell proliferation and stimulate the formation of the blood-testis barrier [19], both indicators of

Sertoli cell maturation. In order to determine if these results could be recapitulated in vivo, neonatal animals were treated with either RA or vehicle from 5-8 dpp and euthanized 24 hours following 4 hours of EdU incorporation. The total number or Sertoli cells, as well as the number of EdU-positive Sertoli cells per round tubule were counted (Figure 7). The vehicle treated animals had an average of 43.1 Sertoli cells and 0.82 EdU-positive Sertoli cells per round tubule.

The RA treated animals had significantly less Sertoli cells and EdU-positive Sertoli cells: 39.5 and 0.31, respectively.

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Discussion

While there has been significant work regarding the generation of testicular RA and the effects of its absence on spermatogenesis, there has been comparatively little work regarding the degradation of this molecule or the consequences of high testicular RA levels. This study focused on providing comprehensive postnatal CYP26 localization within the different testicular cell types, as well as the consequences of high testicular RA.

The CYP26 family of enzymes is responsible for the degradation of RA into inert metabolites [9], yet the protein localization of these enzymes has not been thoroughly investigated in the postnatal testis. Previous studies have reported Cyp26a1 and Cyp26b1 transcripts localized to the PTM cells [13]. CYP26B1 protein has also been localized to the

PTM cells [36]. The data presented in both Figures 2 and 3 agree with this data. Additionally,

CYP26A1 also showed localization to Sertoli cells throughout neonatal testicular development into adulthood. Interestingly, no phenotype was observed when Cyp26a1 was ablated from

Sertoli cells [24], despite being detectable via immunohistochemistry in this cell type. A mild phenotype was observed when Cyp26b1 was deleted from Sertoli cells [24, 25]. This data indicates that although Cyp26b1 was not detected via immunohistochemistry, it is obviously present within Sertoli cells. These knockout models have also highlighted the importance of

CYP26B1; only animals with a mutation in Cyp26b1, either in Sertoli cells or germ cells – or both – showed any spermatogenic defects [14]. The immunohistochemical data presented here, and elsewhere [13], suggests that CYP26 activity within the seminiferous tubules is less important than within the PTM cells. One possible function of the CYP26 enzymes in the PTMs is to provide a metabolic barrier for exogenous RA entering the seminiferous tubules, protecting

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the RA sensitive germ cells. Ablating CYP26 enzymes from the PTM cells will be important for fully understanding the importance of RA degradation in spermatogenesis.

In addition to providing information regarding protein localization, the CYP26 immunohistochemistry provides insights into how the spermatogenic RA pulse is regulated.

Data presented in chapter two provided strong evidence that enzymes responsible for RA synthesis are not for the pulse. Another hypothesis explaining the RA pulse would be the stage- specific regulation of enzymes catalyzing RA degradation, i.e. the CYP26 enzymes. Because there was no stage-specific expression of either Cyp26a1 or Cyp26b1 transcript [13, 14] or protein (Figure 3), it is not likely that RA degradation is variable across the spermatogenic cycle.

Transcript expression suggests that RAL availability is directly responsible for RA pulsatility

[14]. Specifically, Rdh10 and Dhrs4, both of which are known to impact RAL availability, are variably expressed across the spermatogenic cycle [14]. Lrat, responsible for retinoid storage which impacts RAL availability, is also regulated in a stage-specific manner [14].

The inhibition of the CYP26 enzymes, via TAL, is available clinically for the treatment of dermatological diseases such as acne and psoriasis [15]. While the effects of TAL treatment on many tissues, such as the kidney and liver, have been reported [16], the effects on the testis are unknown. The data presented here shows serum TAL levels, testicular RA levels, and testicular Cyp26a1 expression all increase, but the variability prevents statistical significance from being achieved. This variability is likely due to two factors. The first is the extremely quick clearance of TAL from the serum. The second factor is that the increase in testicular RA is going to cause a localized increase in Cyp26a1 expression. Cyp26a1 is known to be regulated by

RA [17], and this negative feedback loop could be partially responsible for the observed variability. TAL treatment in the adults resulted in precocious spermatogonial differentiation.

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This phenotype reciprocated the testicular response to exogenous RA in the adult [35].

Interestingly, this datum shows that raising endogenous RA levels is sufficient to force spermatogonial differentiation, and CYP26 activity is vital for preventing this precocious differentiation event. In the neonatal animal, TAL treatment was able to synchronize spermatogenesis, again similar to the phenotype observed in animals treated with exogenous RA

[17]. This data suggests that increases in endogenous RA are able to synchronize spermatogenesis; i.e. super-physiological doses used with exogenous RA treatment are not required to drive synchronous spermatogenesis. Sertoli and germ cell-specific knockouts of the

CYP26 enzymes do not reciprocate this phenotype [24, 25], indicating that the activity of these enzymes in other cell types, likely the PTMs, is responsible for preventing premature differentiation, though further studies will need to be conducted to verify this.

In addition to misregulation of spermatogonial differentiation, high RA levels cause a loss of spermatocytes in Podarcis sicula [20]. Data presented in the previous chapter also showed that reduced RA levels increase recombination rates. Taken together, these data suggest that RA is playing a role in recombination. After treating synchronized testes with exogenous

RA, recombination rates were shown to be reduced by 0.5 MLH1 foci/cell. This is a modest decrease but it does provide further evidence to suggest that RA is playing a role in the meiotic process.

Sertoli cell maturation has been suggested to be adversely affected by exogenous RA. In vitro, immature rat Sertoli cells were shown to prematurely cease proliferation and form tight junctions [19]: both hallmarks of mature Sertoli cells. When mice were treated in vivo with exogenous RA, there were not only less Sertoli cells present in each tubule, but there was a

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reduction in the number of EdU incorporating Sertoli cells. Both of these data suggest that there is a reduction in Sertoli cell proliferation in animals exposed to increased testicular RA levels.

The data presented here regarding precocious spermatogonial differentiation, lowered recombination rates, and premature Sertoli cell maturation have the potential to be clinically relevant. TAL is currently being used to treat dermatological diseases, such as acne [15], and is known to increase RA levels in various tissues [16], including the testis (Figure 4). These data would suggest that treatment of pubertal and prepubertal individuals with TAL could have adverse effects on testicular development. For example, treatment pubertal boys suffering from acne with TAL could raise testicular RA, which might cause premature cessation of Sertoli cell proliferation. A reduction in Sertoli cells would have an effect on testicular size [37, 38] and could potentially have adverse effects on fertility that would carry into adulthood.

Similar to low testicular RA, high levels of testicular RA has adverse effects on several spermatogenic processes. Here we show that increased testicular RA causes precocious spermatogonial differentiation in the adult and neonate, a decrease in meiotic recombination, and a decrease in Sertoli cell proliferation. It will be important for future studies to investigate the full extent of TAL treatment on the testis. It is also important to determine if, clinically, TAL is having any impact on male fertility, particular in pubertal patients.

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AKOWLDGEMENTS

The authors would like to thank the Isoherranen and Hassold-Hunt laboratories for their technical assistance.

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murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during

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26. Topletz AR, Thatcher JE, Zelter A, Lutz JD, Tay S, Nelson WL, Isoherranen N. Comparison

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hydroxylases. Biochem Pharmacol 2012; 83:149-163.

27. Thatcher JE, Zelter A, Isoherranen N. The relative importance of CYP26A1 in hepatic

clearance of all-trans retinoic acid. Biochem Pharmacol 2010; 80:903-912.

28. Hogarth CA, Griswold MD. Immunohistochemical approaches for the study of

spermatogenesis. Methods Mol Biol 2013; 927:309-320.

29. O'Shaughnessy PJ, Monteiro A, Abel M. Testicular development in mice lacking receptors

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30. Russell LD, Ettlin RA, Sinha Hikim AD, E.P. C. Histological and Histopathological

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31. Arnold SL, Kent T, Hogarth CA, Griswold MD, Amory JK, Isoherranen N. Pharmacological

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32. van Beek ME, Meistrich ML. Stage-synchronized seminiferous epithelium in rats after

manipulation of retinol levels. Biol Reprod 1991; 45:235-244.

33. Siiteri JE, Karl AF, Linder CC, Griswold MD. Testicular synchrony: Evaluation and analysis

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34. Peters AH, Plug AW, van Vugt MJ, de Boer P. A drying-down technique for the spreading of

mammalian meiocytes from the male and female germline. Chromosome Res 1997; 5:66-68.

35. Endo T, Romer KA, Anderson EL, Baltus AE, De Rooij DG, Page DC. Periodic retinoic

acid-STRA8 signaling intersects with periodic germ-cell competencies regulate

spermatogenesis. Proc Natl Acad Sci U S A 2015; ahead of print Apr 2015.

36. Wu JW, Wang RY, Guo QS, Xu C. Expression of the retinoic acid-metabolizing enzymes

RALDH2 and CYP26b1 during mouse postnatal testis development. Asian J Androl 2008;

10:569-576.

37. Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hormone is required for

ovarian follicle maturation but not male fertility. Nat Genet 1997; 15:201-204.

38. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation

factor-9 is required during early ovarian folliculogenesis. Nature 1996; 383:531-535.

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FIGURES

Figure 1 Polyclonal antibodies show specificity to CYP26 enzymes.

The figure shows representative immunoblots using antibodies specific to CYP26A1 and

CYP26B1. A single band was observed for each of the two antibodies used. No antibody was detected in the negative control.

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Figure 2: CYP26 localization in the neonatal murine testis

Images are representative murine testicular cross-sections displaying immunohistochemical analysis of CYP26 localization at various neonatal ages. CYP26A1 is represented in A,C, E, G,

I, and K for 0, 5, 10, 15, 20, and 30 dpp, respectively. CYP26B1 is represented in B, D, F, H, J, and L for the same time points. Negative controls are shown in M and N. Brown staining indicates an immunopositive reaction. Arrows indicate immunopositive cells while the respective colors indicate the following cell types: Red – Sertoli cells, Yellow –

Leydig/Interstitial cells, Orange- Peritubular myoid cells. Scale bars represent 100 μm.

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Figure 3. CYP26 enzymes locate predominantly to PTM cells in the adult murine testis.

Images are representative adult murine testicular cross-sections displaying immunohistochemical analysis of CYP26 localization. CYP26A1 and B1 localization can be seen in A and B, respectively. Negative controls for each assay are shown in the picture insert. Brown staining indicates an immunopositive reaction. Arrows indicate immunopositive cells while the respective colors indicate the following cell types: Red – Sertoli cells, Yellow –

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Leydig/Interstitial cells, Dark Blue – Spermatid, Orange – Peritubular myoid cells. Scale bars represent 100 μm. A summary of the adult localization data is represented in C. A solid line underneath a cell indicates that cell is immunopositive for the respective CYP26. CYP26A1 and

B1 are represented by blue and yellow, respectively. Stage diagram adapted from Hogarth and

Griswold, 2010.

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Figure 4. Talarozole is cleared very quickly from the serum

This graph represents serum TAL levels (y-axis) over time following a single TAL treatment (x- axis). Scale bars represent the SEM. No statistical significance was achieved at any point.

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Figure 5 Talarozole treatment of adult male mice results in misregulation of spermatogonial differentiation.

A) The graph represents testicular RA concentrations (y-axis) over time following 3 days of TAL treatment (x-axis). B) Cyp26 transcript levels were measured (y-axis) via RT-PCR over time following 3 days of TAL treatment (x-axis). C-D) Representative histological cross-sections of adult testis tubules following 3 days of vehicle, RA, or TAL treatment, respectively (n=3 for

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each treatment). Brown staining is indicative of STRA8 positive cells. F) STRA8 positive spermatogonia were quantified from histological cross-sections of animals treated for 3 days with vehicle, RA, or TAL. For all graphs, vehicle, RA, and TAL treated animals are represented by blue, red, and green respectively. (* p < 0.05, ** p < 0.01)

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Figure 6 Neonatal exposure to talarozole drives synchronous spermatogenesis

Representative images of histological cross-sections from the testis of animals treated with either vehicle (A) or TAL (B). Brown staining is indicative of STRA8 positive cells. The average synchrony factor was determined (C) for animals treated with vehicle (Blue) and TAL (Green).

The graph in D represents the percentage of tubules (y-axis) present at each stage (x-axis).

Roman numerals are used to label spermatogenic stages and the letter “D” in the x-axis of Figure

D is labeling disrupted (unscorable) tubules. (* p < 0.05)

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Figure 7 Exogenous neonatal RA treatment reduces Sertoli cell proliferation

Representative histological testicular cross-sections from 9 dpp mice treated for 4 days with RA staining for EdU (green) and SOX9 (red). DAPI was used as a counter stain (blue). Average

SOX9-positive cells per round tuble (I) and EdU/SOX9-positive cells per round tubule (J) were quantified for both vehicle (blue bars) and RA (red bars) treated animals (n=3). (*p < 0.05)

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TABLES

Table 1: Exogenous RA treatment reduces the recombination rate in testes with synchronized spermatogenesis

MLH1 foci/cell e0 e1 e2 e3

Vehicle 23.15 ± 0.26 1.05% 75.93% 22.74% 0.14% RA 22.46 ± 0.23* 1.16% 79.53% 19.26% 0.05%

MLH1 foci/cell were quantified and averaged for animals with synchronous spermatogenesis treated with either vehicle or RA. e0, e1, e2, and e3 denote the percentage of chromosomes containing 0, 1, 2, and 3 exchanges, represented by MLH1. A student t-test was used to determine if there was a statistically significant difference in recombination rate between control and RA treated animals (* p = 0.050).

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CHAPTER 4

CONCLUSIONS AND FUTURE DIRECTION

Conclusions

It has been well documented that retinoic acid (RA) is important for the production of sperm [1]. The overall regulation of the synthesis and degradation of this critical molecule within the testis, however, is not fully understood. The data presented herein provides a better understanding of the enzymes directly responsible for the availability of RA and the consequences of transient inhibition of these enzymes. Together these data provide a more comprehensive understanding of testicular RA regulation and the effects of aberrant testicular

RA levels on spermatogenesis.

Testicular RA levels are heavily influenced by how retinoids are metabolized. Vitamin A is ingested via the diet, and upon reaching its target tissue, it is converted to RA through two enzymatic reactions [2]. The enzymes directly responsible for the synthesis of RA are the

ALDH family of enzymes [2]. RA then either signals through the appropriate signaling pathways, or is degraded into inert metabolites by the CYP26 family of enzymes [2]. In utero,

RA gradients are established based on the localization of the ALDH and CYP26 enzymes [3-6], but it is not known if this is the case in the postnatal testis. There has been very little data collected regarding the postnatal testicular localization of either the ALDHs or CYP26s. The work that has been performed predominately focuses on mRNA localization, which provides absolutely no information regarding enzyme activity, and reports have been contradictory and incomplete [7-10]. The data presented in chapters 2 and 3 is the most comprehensive analysis of postnatal testicular localization for both of these enzyme families. It is clear that the individual

ALDH isozyme localization patterns differ from one another while the CYP26 localization patterns are much more uniform. Specifically, ALDH1A1 localizes predominantly to Sertoli cells while ALDH1A2 and ALDH1A3 localize to spermatocytes and spermatids. While the role

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for RA synthesis in Sertoli cells has been investigated [11], the reason for RA synthesis in the meiotic and post-meiotic cells remains unclear. It has been hypothesized that RA synthesis in the Sertoli cell is required during the first spermatogenic wave, but the RA required for spermatogonial differentiation of all subsequent waves is synthesized in the meiotic cells [8, 12].

The immunohistochemistry, particularly ALDH1A2, presented in Figure 1 and 2 of chapter 2 support this hypothesis. While CYP26A1 expression was noted in Sertoli cells, the PTM cells exhibited the strongest CYP26 staining for both isozymes (Figure 2 and 3 of chapter 2), indicating a yet unknown role for RA degradation in these cells, perhaps acting as a metabolic barrier preventing RA from entering into the seminiferous tubule.

The localization of these enzymes can also provide information regarding the regulation of RA across the spermatogenic cycle. For example, mRNA localization studies have led the prediction that RA is important for embryonic tissue patterning and organogenesis [3-6]. There are several lines of evidence that show RA availability is regulated in a variable manner within the testis. For example, STRA6 and STRA8, both known to be under the direct regulation of

RA, are present in a stage-specific manner [13-15]. More directly, Hogarth et al. has shown that

RA levels vary significantly across the spermatogenic cycle, with peak RA levels being present at Stages VIII and IX [14]. While the manner in which this pulse is regulated is not currently known, it has been hypothesized that the ALDH enzymes are responsible for the stage-specific availability of RA [7, 10, 15]. It is also possible that CYP26 enzymes could degrade RA in a stage-specific manner, causing variable RA levels.

The data presented here provides evidence that the ALDH and CYP26 enzymes are not regulating the RA pulse. Previous reports of stage-specific mRNA expression show that enzymes in both families do not drastically change across the spermatogenic cycle [14]. This is

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confirmed in the protein localization data for both ALDH and CYP26 enzymes. While exhibiting different cell localization patterns, all of these enzymes were present in every stage.

To extend on this, HPLC MS/MS was utilized to quantify ALDH isozyme levels, as well as total

ALDH activity on testes with synchronized spermatogenesis. The results from both of these assays showed no changes in ALDH1A1 or ALDH1A2 levels across the cycle, and no changes associated with total ALDH activity. These data, together, provide strong evidence that ALDHs are not responsible for pulsatile changes in testicular RA levels. Additionally, the CYP26 localization suggests that RA degradation is not changing across the spermatogenic cycle, although further studies could be performed to confirm this assertion. Specifically, CYP26 protein quantification and activity assays across the spermatogenic cycle would provide definitive evidence regarding the contribution of RA degradation to the pulse.

ALDH and CYP26 enzyme families are directly responsible for testicular RA availability. There are studies showing that aberrant testicular RA levels have adverse effects on spermatogenesis. Lowered testicular RA levels, whether via dietary restriction or chemical inhibition, result in the loss of sperm production [16-20]. Lowered RA availability is also thought to have an adverse effect on spermiation and blood-testis barrier permeability [11, 20,

21]. Conversely, there is evidence to suggest that increased testicular RA causes misregulation of Leydig cells, Sertoli cells, spermatogonia, and spermatocytes [8, 12, 15, 22-24]. Clinically, misregulation of testicular RA, either too high or too low, could be a cause for idiopathic male subfertility or infertility. The data presented here supports the idea that aberrant testicular RA causes problems in various spermatogenic processes.

Data presented in both chapters 2 and 3 show that altered testicular RA levels have an effect on meiotic recombination. When adult animals were treated with WIN 18,446, a potent

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ALDH inhibitor [25], there was a significant increase in meiotic recombination. The converse was true when animals were treated with exogenous RA, i.e. there was a decrease in recombination rates in animals treated with RA compared to control animals. Additionally, animals treated with WIN 18,446 showed an increase in meiotic defects compared to control animals. While STRA8 is known to be important for early meiosis [26, 27], this is the first evidence to suggest a role for RA in meiotic recombination.

Proper Sertoli cell function has been suggested to be under the regulation of RA.

Specifically, blood-testis barrier (BTB) specific genes were misregulated in the vitamin A deficient testis [28]. Additionally, blocking RAR signaling in the Sertoli cell disrupted BTB permeability [29]. Conversely, immature rat Sertoli cells cultured with exogenous RA prematurely matured [23]. The data presented here shows that BTB permeability increases in animals with a low testicular RA environment. Additionally, neonatal exposure to exogenous

RA decreases Sertoli cell proliferation. Taken together, these data show that normal testicular

RA levels are required for proper Sertoli cell function.

Finally, RA has been well studied in terms of spermatogonial differentiation. The A-A1 transition is halted in RA deficient animals and reintroduction of RA results in immediate spermatogonial differentiation (reviewed in [30]). Additionally, exogenous RA in the adult caused precocious spermatogonial differentiation [15], while exogenous RA in the neonate stimulated synchronous spermatogenesis [8, 12]. The data presented here shows that treatment with TAL is sufficient to recapitulate the phenotypes observed with exogenous RA. These data show that, not only is endogenous RA sufficient to cause precocious spermatogonial differentiation, but CYP26 activity is also playing an active role in opposing this effect. Which cell types are playing a role in this suppressive role is not yet clear, but based on the

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immunohistochemical data presented in chapter 3, I hypothesize that exogenous RA from outside of the seminiferous tubule is degraded by CYP26 activity in the PTM cells, preventing premature germ cell differentiation. More work in this area will be required to verify this hypothesis.

Additionally, super-physiological levels of RA are not required to induce synchronous spermatogenesis, something that should be noted when treating pubertal boys with TAL.

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Future Directions

The data presented here provides many insights, upon which future research should be performed. While the role of RA in spermatogenesis has been well studied, there are still plenty of unknowns left to investigate. One of the most interesting questions is what regulates the observed pulse of RA in the testis. The data here shows that it is likely not the ALDH or the

CYP26 families of enzymes, although protein quantification and enzyme activity assays performed in a stage-specific manner on the CYP26 enzymes would be beneficial to verify that

RA pulsatility is happening independent of RA degradation. Perhaps the answer lies in the regulation of the rate limiting step in the RA synthesis pathway: the conversion of retinol to retinaldehyde (RAL), the substrate for ALDH enzymes [31]. Two genes known to regulate the availability of RAL, Rdh10 and Dhrs4, are present in a stage-specific manner [14]. Thus far, however, no testicular localization for either enzyme has been reported. It should be noted that

Sertoli and Sertoli-germ cell ablations of RDH10 resulted in delayed spermatogenesis [19], indicating its presence in these cell types neonatally. Determining the localization patterns of enzymes responsible for RAL availability, specifically RDH10 and DHRS4, is crucial in determining how the RA pulse is regulated. Additionally, stage-specific measurements of both retinol and RAL will be important in determining which metabolites in the vitamin A metabolism pathway are being regulated in stage-specific manner. This would provide insight into which enzymes are responsible for this phenomenon.

Other important areas of future study relate to altered testicular RA levels. There is a growing body of evidence, including data presented here, showing altered testicular RA levels have adverse effects on normal spermatogenesis. One key area that needs to be addressed is whether aberrant testicular RA levels are seen in human populations, particularly those who are

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unable to conceive a child. Determining an easily assayed biomarker of testicular RA could be a vital tool towards the diagnosis of idiopathic male infertility. It will also be important to determine all of the effects of lowered testicular RA on spermatogenesis. For example, one area that has not been investigated is the role of RA in spermiogenesis. The RNA sequencing data from the testes of animals treated with WIN 18,446 showed a decrease in genes associated with proper spermatid function, among others. This, in conjunction with a study showing that ablation of Rara causes a delay in spermatid maturation during Stage VIII [32], suggests a novel role for RA in proper spermatid differentiation.

The meiotic data presented here suggests a role for RA in this process as well. While

STRA8, known to be important for early meiosis [26, 27], has long been known to be under the regulation of RA [33], the observed change of recombination rate in both high and low testicular

RA environments is novel. The mechanism behind these altered recombination rates is completely unknown. Interestingly, RA plays important roles for spermatogonial differentiation,

BTB reorganization, meiotic initiation, and spermatid release, all of which take place during the same two spermatogenic stages: VII and VIII. Is RA signaling occurring in pachytene spermatocytes during these stages as well? Thus far, there have not been any reports of RARs or

RXRs present in spermatocytes. If signaling is occurring, what specifically is it regulating? If not, how are these cells escaping the pulse of RA that appears to affect all other cell types within the seminiferous tubules during this stage of the cycle? Spermatocyte specific ablation of RA signaling machinery could be instrumental for addressing these questions.

The effects of altered testicular RA levels on Sertoli cells are also not fully understood, though evidence suggests that both high and low RA has consequences. Inhibiting RA signaling in the Sertoli cell [29] or lowering testicular RA levels both increased BTB permeability. If the

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perturbation of vitamin A metabolism/signaling pathway is to be pursued as a viable approach towards an effective male contraceptive, as it currently is, the long term effects of these perturbations must be thoroughly investigated. TAL too, is being used clinically as a therapeutic for dermatological disease [34], yet the reproductive consequences of these treatments is currently unknown. The data presented here is particularly concerning. TAL raises RA levels in various organs [35]. In the testis, TAL exposure resulted in misregulation of spermatogonia.

Increased testicular RA levels decreased Sertoli cell proliferation and lowered total Sertoli cell numbers. Reduced numbers of Sertoli cells has been linked to decreased testes size [36, 37], which would lead to reduced sperm output and lower fertility. Given that TAL is used to treat acne, among other dermatological diseases, treating pubertal boys should be cause for concern until further experiments are performed to assess the long term reproductive consequences of

TAL treatment.

It is evident that RA is absolutely essential for spermatogenesis. It is equally evident that we have only begun to elucidate RA’s role in this process. The results presented in this work clearly show the need for further research into the role of RA during mammalian spermatogenesis, particularly how this molecule is regulated in the context of the spermatogenic cycle and how aberrant testicular RA levels adversely affect spermatogenesis, especially in humans. Because RA is a key regulator of spermatogenesis, understanding both the synthesis and degradation of this molecule, as well the reproductive consequences if these processes are not executed properly, will greatly improve our understanding of testicular biology. This dissertation represents a small step towards that goal.

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