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Home , CENPA, Centromere, Chromosome 13, Chromosome 15, Chromosome 3, Chromosome segregation

INVESTIGATION OF FACTORS AFFECTING FERTILITY:

SEGREGATION ERRORS AND ENVIRONMENTAL TOXINS

by

JODI M. JACKSON

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Thesis Advisor: Dr. Patricia A. Hunt

Department of

CASE WESTERN RESERVE UNIVERSITY

August, 2007

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______

candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

______

______

______

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein.

I dedicate this dissertation to the loving memory of my grandmother, Margaret Franz. Her unwavering conviction that education opens doors, coupled with her sacrifice to ensure her family had opportunities not available to her, propelled me far beyond where my own endurance surely would have failed.

iv Table of Contents

Dedication………………………………………………………………………………..iv

Table of Contents………………………………………………………………………...v

List of Tables……………………………………………………………………………..x

List of Figures...... xi

Abstract…………...... xii

Chapter 1: Introduction and Research Aims

Introduction…………………………………………………………………...... 1

Why and Environmental Toxins?...... 1

What Happens When Chromosome Segregation Goes Wrong?...... 2

The BALB/cWt ………………………………………….3

Chromosome Segregation in Spo13 Deficient S. cerevisiae……………...4

What is a ?...... 5

Centromere Sequence…………………...………………...………6

Centromere ………………………………………….……9

What is a Good Strategy to Study the Centomere?...... 13

What is Known About the Y Centromere?...... 13

Environmental Toxins and Reproduction……………………….16

Is the Incidence of Human Fertility Increasing?...... 16

Impacts of Environmental Toxins on Male Infertility…………………...17

Medical Exposure………………………………………………..17

Occupational Exposure…………………………………………..18

v Day-to-Day Exposure……………………………………………18

Impacts of Environmental Toxins on Infertility………………...22

Medical Exposure………………………………………………..22

Occupational Exposure…………………………………………..22

Day-to-Day Exposure……………………………………………23

What are Quaternary Ammonium Compounds?...... 24

Chapter 2: The mouse A/HeJ Y chromosome: Another good Y gone bad………..28

Abstract………………………………………………………………………….29

Introduction……………………………………………………………………...30

Methods and Materials…………………………………………………………..32

Mice…………………………………………………………….………..32

Southern Blot Analysis………………………………………….……….33

Fetal Analysis and Chromosome Preparations…………………..34

Fluorescent In Situ Hybridization Studies……………………………….35

Y Chromosome Mapping………………………………………..35

Aneuploidy Analysis…………………………………………….36

Meiotic Preparations…………………………………………………….36

Diakinesis and II…………………………………….36

Pachytene………………………………………………………...37

Results…………………………………………………………………………...37

Hermaphroditism is a feature of the A/HeJ strain……………………….37

Hermaphroditism is a property of the A/HeJ Y chromosome…………...38

vi The YA/HeJ and YWt are different on the sequence level….39

The YA/HeJ chromosome is prone to mitotic mis-segregation……………41

The YA/HeJ centromere exhibits morphological aberrations……………...42

The YA/HeJ chromosome segregates faithfully in ………………..46

The structural integrity of the YA/HeJ chromosome………………………48

Discussion………………………………………………………………………..51

Structural evidence that the YA/HeJ and YWt chromosomes are different...52

Behavioral differences between the YA/HeJ and YWt chromosomes……...53

Why does the YA/HeJ cause disturbances in ?...... 53

Acknowledgements………………………………………………………………58

Chapter 3: Disinfectants and Dysfunction: Solving the Mystery…………………..59

Abstract…………………………………………………………………………..60

Introduction………………………………………………………………………61

Methods and Materials…………………………………………………………...62

Breeding Protocols……………………………………………………….62

Superovulation…………………………………………………………...62

Methanol Cage Extractions………………………………………………62

Analytical Methods………………………………………………………63

Results…………………………………………………………………………....64

Problems in a new breeding colony are immediately apparent………….64

Ruling out the obvious candidates……………………………………….65

New research studies reveal continued reproductive disturbances………67

vii Are we victims of our own research?...... 70

Discontinued use of the disinfectant but persistence of the chemical

compounds……………………………………………………………….72

Inadvertent recontamination……………………………………………..73

Discussion………………………………………………………………………..77

Chapter 4: Summary and Future Directions………………..……………………….80

Summary…………………………………………………………………………80

The Mouse A/HeJ Y Chromosome: Another Good Y Gone Bad……….80

The A/HeJ Y Chromosome Induces Hermaphroditism………… 80

Comparison on A/HeJ and BALB/cWt…………………………..81

Sequence…………………………………………………81

Segregation Behavior…………………………………….82

Structural Analysis of the YA/HeJ Chromosome………………….83

Morphological Aberration of the A/HeJ Y Centromere…………83

Disinfectants and Dysfunction: Solving the Mystery…………………...84

Breeding Problems in the New Colony………………………….84

Changing the Environment………………………………………84

Reproductive Problems Continue………………………………..85

Identifying the Source of the Reproductive Disturbances……….85

Eliminating the Quaternary Ammonium Disinfectant…………...86

Accidental Recontamination……………………………………..87

Future Directions………………………………………………………...88

viii Studies of the A/HeJ Y Chromosome……………………………88

Centromere……………………………..………………...88

Sexual Differentiation…………………….……...………89

Epigenetic Analysis………………………...……………91

Analysis of the Effect of Quaternary Ammonium Compounds on

Reproduction……………………………………………..92

Bibliography…………………………………………………………………………….95

ix List of Tables

Chapter 1

Table 1.1 Examples of proteins that localize to the centromere and and their

functions…………….……………………………………………………………11

Table 1.2 Examples of studies of the impact of chemical exposure on human fertility...21

Table 1.3 Examples of common quaternary ammonium compounds and the types of

products typically containing each one………………………………………….26

Chapter 2

Table 2.1 Analysis of Y chromosome hyperploidy and gaps…………………….…...... 42

Table 2.2 Meiotic analysis in C57BL/6J-YA/HeJ males…………………………….…….47

x List of Figures

Chapter 1

Figure 1.2 Regions of the centromere and kinetochore………………………………....12

Chapter 2

Figure 2.1 Southern analysis of Bkm repeats in genomic DNA………………………...40

Figure 2.2 The frequency of Y chromosome hyperploidy and of gaps

are not correlated in individual C57BL/6-YA/HeJ ……………………44

Figure 2.3 Representative images of YA/HeJ chromosome morphological aberrations

observed during analysis of A/HeJ Y chromosome in liver and fibroblast

cells…………………………………………………………………………..45

Figure 2.4 Schematic showing the normal localization and a brief description of the eight

FISH probes used in the structural analysis of the A/HeJ Y chromosome…..49

Figure 2.5 Structural analysis of the A/HeJ Y chromosome……………………………50

Chapter 3

Figure 3.1 A morphologically abnormal 18.5 day A/J fetus…………………….………66

Figure 3.2 Pregnancy rates in young (6-8 week) virgin C57BL/6J ………….…68

Figure 3.3 Cage extracts reveal contamination with quaternary ammonium

compounds……………………………………………………………….…..73

Figure 3.4 Quaternary ammonium contaminants are transferred during cage washing...76

xi Investigation of Factors Affecting Fertility: Chromosome Segregation Errors and

Environmental Toxins

Abstract

by

JODI M. JACKSON

If a cannot propagate, it cannot survive. Human fertility is thought to be

declining, with a myriad of possible causes. The objective of this dissertation was to investigate two factors that affect fertility: chromosome segregation errors and

environmental toxins. affects 10-30% of human conceptions and is the

leading genetic cause of pregnancy loss and mental retardation. Accordingly, the

etiology of chromosome segregation errors has long been studied. Conversely, while

examples of environmental toxins impacting fertility can be found throughout the

literature, this field is only recently becoming a major focus of research. The studies

done for this dissertation were done independently from one another and are presented

here separately.

The observation of hermaphroditic mice on the inbred strain A/HeJ prompted the

investigation of the YA/HeJ chromosome. The YA/HeJ chromosome was transferred to the

C57BL/6J background and the hermaphroditism trait segregated with it, indicating the

two are linked. We found the YA/HeJ chromosome to be prone to missegregation,

however, we did not find evidence that segregation errors cause the disturbances in

xii sexual differentiation. Interestingly, we frequently observed a gap between YA/HeJ sister , indicating inappropriate centromere function. The mouse Y centromere is near the -determining and we hypothesize that a (s) in this region affects both the YA/HeJ centromere (causing the gaps) and expression (causing the ). The YA/HeJ chromosome serves as a model to study centromere structure and function and, ultimately, what makes a chromosome prone to segregation error.

After our laboratory relocated from Case Western Reserve University in

Cleveland, Ohio, to Washington State University in Pullman, Washington, we experienced a significant decline in the reproductive fitness of our mouse colony.

Pregnancy rates, egg and quality, fetal development and pup survival were all negatively impacted. After investigating numerous environmental variables, we determined it was the facility-wide use of a quaternary ammonium disinfectant that was negatively affecting our mouse breeding colony. By eliminating the disinfectant from the facility and increasing the stringency of the automated cage washer, we were able to greatly improve the breeding performance of the mouse colony.

xiii Chapter 1: Introduction and Research Aims

Introduction

The ability to propagate is the most critical biological function of a species, and

yet, are exceedingly poor at it. According to the Centers for Disease Control

(CDC), approximately 12% of American women of child-bearing age have sought infertility treatment and 7% of couples report trying to conceive unsuccessfully for at least a year. Beginning in 1978, persons incapable of becoming pregnant through natural means have been able to seek treatment through Assisted Reproductive Technologies

(ART), such as hormone stimulation, in vitro fertilization (IVF) and intracytoplasmic injection (ICSI). In 2004 (the most recent data available), approximately 128,000

ART cycles were initiated (CDC 2006). At an average cost of $12,000 per cycle, this equals approximately $1.5 billion a year being spent in American fertility clinics (Luoma

2005). While there are undoubtedly multiple causes of impaired fertility, this dissertation will focus on two: chromosome segregation errors and environmental toxins.

Why Chromosome Segregation and Environmental Toxins?

The studies originally outlined in my thesis proposal focused solely on mitotic chromosome segregation errors using the mouse as a model system. As is not uncommon in science, however, the research deviated from this path. After our laboratory moved from Case Western Reserve University to Washington State University, we experienced a drastic decline in the overall reproductive fitness in our mouse colony. Many experiments necessary for the chromosome segregation studies were no longer possible

1 and consequently, separate research was initiated to identify and eliminate the source of reproductive impairment. After methodically changing environmental conditions in an attempt to mimic those at Case, we were surprised to discover that the disinfectant being

used in the facility was responsible for the changes in the mice. The phenotypic

consequences of this exposure included a significant decline in the pregnancy rate, unusual developmental defects and late fetal loss, suggesting an impact on a variety of tissues and pathways. Thus, this thesis encompasses two separate studies: the originally

proposed studies of mitotic chromosome segregation and the results of studies designed

to identify and eliminate the reproductive disturbance at WSU. While both set of studies

involve fertility, they will be considered individually, starting with chromosome

segregation.

What Happens When Chromosome Segregation Goes Wrong?

Aneuploidy, the loss or gain of a chromosome, occurs in varying frequency in all

. Most model organisms exhibit low spontaneous rates of meiotic aneuploidy,

e.g., missegregation occurs in 0.01% of , ~0.02% of

the time in the female germ line of melanogaster and in 1-2% of fertilized

eggs in Mus musculus. By comparison, humans have alarmingly high rates of

aneuploidy: 1-2% in sperm, ~20% in oocytes and 35% of spontaneous abortions

(reviewed in Hassold and Hunt 2001). Why humans have this marked increase in errors

is not yet known. Interestingly, however, the risk of missegregation is not equal for all

human chromosomes, but appears to be chromosome-specific. For example, 16

is the most commonly identified autosomal aneuploidy, while conversely, 1 and

2 19 are rare, if not non-existent (Hassold and Jacobs 1984). This variation could be interpreted in one of two ways: either these, and presumably other, chromosomes

missegregate at different frequencies, or equal numbers of errors occur, but are not

uniformly eliminated during pregnancy. The fact that certain trisomies such as 13, 18

and 21 are more frequently observed in liveborns than , yet are observed less

often in clinically recognized pregnancies, argues that there truly are differences in

occurrence as well as differences in in utero selection (Hassold 1986).

While chromosome-specific variation in has been best

characterized in humans, nondisjunction-prone chromosomes also have been reported in

model organisms. Below we consider two examples, one in mouse and one in yeast.

The BALB/cWt Y Chromosome:

The Y chromosome from the inbred strain BALB/cWt serves as an example of an

error-prone mouse chromosome. BALB/cWt was originally characterized by Whitten

(1975) as having a high (3.0%) incidence of hermaphroditism, a low (38% male) and a high incidence of XO females. On the basis of these observations, Whitten hypothesized that disturbances in testis determination were caused by nondisjunction of the Y chromosome. Beamer et al. (1978) compared fetal liver (specifically,

the number of cells either bearing or lacking a Y chromosome) with the proportion of

ovarian tissue in individual XY hermaphrodites and noted that the two were positively

correlated. This prompted them to suggest that the proportion of XO to Y-bearing cells

determined whether the differentiating gonad developed along female or male lines. In a subsequent analysis by this group, Eicher et al. (1980), made two observations: 1)

3 ovarian tissue would develop if an animal had between 24-44% XO liver cells and 2) the

majority of Y missegregation must occur before the hepatic and gonadal cells

differentiated from one another. More recently, work by Bean et al. (2001) demonstrated

that no other BALB/cWt chromosome is prone to missegregation, only the Y

chromosome. Further, they found that the propensity for error was significantly higher

during the first two embryonic cleavage divisions compared to subsequent divisions.

Thus, the BALB/cWt Y chromosome appears to be error-prone early in development,

presumably generating mice for aneuploidy, but then “settles down” later in development and segregates normally.

In Chapter 2, studies of another nondisjunction-prone mouse Y chromosome, the

A/HeJ Y, are summarized. In addition to Y chromosome segregation errors, A/HeJ is similar to BALB/cWt in that there is also an increased incidence of hermaphroditism.

However, unlike the YWt, YA/HeJ missegregation does not appear to not be limited to the

early embryonic divisions and does not appear to be responsible for the observed

hermaphroditism.

Chromosome Segregation in Spo13 Deficient S. cerevisiae:

Chromosome missegregation events are extremely rare in S. cerevisiae.

However, there is evidence that endogenous chromosomes behave differently under meiotic stress. For example, in an analysis of a mutation (Spo13) that results in an

abnormal meiosis with only a single division, Hugerat and Simchen (1993) found that

chromosome segregation was “mixed”. That is, some chromosomes behaved as if they

were in meiosis I (segregating whole chromosomes) while others behaved as if they were

4 in meiosis II (segregating ). Each chromosome studied had a specific

segregation pattern, which they concluded was a product of individual . This hypothesis of centromere-driven segregation is not exclusive to yeast chromosomes, but has been suggested for other missgregation-prone chromosomes. In fact, as discussed in

Chapter 2, the Y chromosomes of both the BALB/cWt and A/HeJ mouse strains appear to have been mutated at/near the centromere, resulting in impaired segregation. But to

understand how a centromere could affect chromosome segregation, it is first necessary

to briefly describe centromere structure and function.

What is a Centromere?

There are multiple definitions for a centromere. Originally, it was defined by its

cytological appearance; i.e., as the narrowest part of the chromosome, sometimes called the “primary constriction”. However, it has at least two other important attributes.

Genetically, it is defined as the area of a chromosome that never recombines during

meiosis; thus, the genetic “identity” of a chromosome is conferred by its centromere.

Functionally it is the area of the chromosome that attaches to the spindle ,

thus allowing the chromosome to segregate during mitotic or meiotic division.

These definitions, however, merely describe a centromere. Despite all the

advances in genetics and chromosome biology, a question that remains is what is a centromere? What are the essential elements? There are two levels of focus to answer this question: sequence and proteins.

5 Centromere Sequence

The DNA sequences that characterize centromeres vary in size and complexity

among organisms, from small “point” centromeres to large regional centromeres

spanning megabases of DNA. For example, in budding yeast such as Saccharomyces

cerevisiae, or C. Glabrata point centromeres may consist of few as 125-150 bp. In

contrast, in the fission yeast S. pombe the centromeric sequence is complex and varies

from between 35-110 kb, while centromeres are larger still

(~400 kb) (Clarke 1998). Further, in higher eukaryotes, centromeres typically contain

megabases of repetitive sequence (reviewed in Malik and Henikoff 2002; reviewed in

Cleveland, Mao et al. 2003). In humans, centromeric regions are composed of a complex

pattern of alpha satellite DNA. Alpha satellite DNA consists of a ~171 bp monomer

which, in the regions flanking the centromere, are arranged as simple repeating units.

However, in the centromere “proper” (i.e., the region that is associated with normal chromosome segregation), alpha satellite DNA is arranged into more complex higher order repeats which may extend for several megabases (Willard 1987; Varmuza, Prideaux

et al. 1988; Willard 1991; Warburton, Haaf et al. 1996; Alexandrov, Kazakov et al. 2001;

Schueler, Higgins et al. 2001; Nusbaum, Mikkelsen et al. 2006).

Much less in known about the structure of mouse centromeres. Nevertheless, like

other higher eukaryotes it is comprised of different types of satellite . Specifically, four kinds of satellite repeats have been identified in mouse centromeric regions: major, a 234 bp monomer; minor, a 120 bp monomer; and the more recently identified MS3 and

MS4, which are 150 bp and 300 bp long, respectively (Horz and Altenburger 1981; Wong and Rattner 1988; Kuznetsova, Prusov et al. 2005). Major and minor satellite DNA were

6 first identified as a part of genomic DNA in 1961 (Kit) and localized to the centromere in

1970 (Pardue and Gall; Cooke, Bazett-Jones et al.). Minor satellite is located in the

center of the centromere, while major is found in the flanking pericentromeric

(Ross, Grafham et al. 2005). Both major and minor satellite repeats are

A+T rich and were isolated through classical cesium chloride gradient centrifugation

studies. In contrast, MS3 and MS4 repeats are G+C rich and were identified through

library screens and computer analysis (Kit 1961; Kuznetsova, Prusov et al.

2005). MS3 and MS4 repeats are intermingled with the other two types of repeats—MS3

with minor and MS4 with major (Kuznetsova, Podgornaya et al. 2006). No specific function has yet been assigned to any of the types of satellite DNA. It has been hypothesized that due to its location and associations, minor satellite may play a role in centromere function (Broccoli, Miller et al. 1990). It is interesting to note, however, that, while present on all other chromosomes, none of the four types of satellite

DNA have been localized to the centromere of the mouse Y chromosome (Pardue and

Gall 1970; Broccoli, Miller et al. 1990).

While both human and mouse centromeres are comprised of satellite repeats, there are at least two types of atypical chromosomes that suggest these sequences are neither necessary nor sufficient for conferring centromere function: dicentric chromosomes and chromosomes containing . Dicentrics are chromosomes that have two centromeres, either as a result of an error in replication or recombination or as a result of a breakage and reunion event. Conceptually, three types of dicentric chromosomes are possible: one containing two centromeres that are copies of one another (e.g., duplicated copies of a centromere from a single ), one

7 containing non-identical centromeres from the same homologue (e.g., one centromere

from one chromosome 13 and one from the other chromosome 13), and one containing

centromeres from non-homologous chromosomes (e.g., one centromere from

chromosome 13 and one from ). All three types of dicentrics have been

identified in humans, with considerable variation in the behavior of the chromosomes in

. However, several observations are of particular importance. First, both

centromeres can be active, but this is not always the case; dicentric X chromosomes

provide examples of both outcomes. Sullivan and Willard (1998) reported dicentric X

chromosomes in which both centromeres are active, while Therman et al. (1986) reported only one active centromere per chromosome. Thus, in the latter case, the cell is somehow able to “choose” which of the two identical sequences is to be silenced. Further, in

Robertsonian translocations carrying centromeres from non-homologous chromosomes, one centromere can be silenced in one cell and active in another (Dewald, Boros et al.

1979); thus, depending on the cellular environment, it can be “out-competed”. Taken together, these observations indicate that a specific sequence may be sufficient, but not necessarily required, for centromeric function.

In contrast to dicentric chromosomes, chromosomes with neocentromeres have functional centromeres in regions lacking the typical centromeric DNA architecture. For example, in humans, neocentromeres form de novo in genomic sequences that do not contain alpha satellite repeats. Typically, they arise on rearranged chromosomes that have lost their endogenous alpha satellite repeats, but occasionally neocentromeres are found on chromosomes that retain alpha satellite sequences. To date, neocentromeres have been observed on 19 of the 24 human chromosomes, and, while there appear to be

8 “hotspots” (areas predisposed to formation), there is no consensus

sequence (reviewed in Warburton 2004). Thus, a specific DNA sequence is not necessary for neocentromere formation. Presumably, since sequence does not confer centromere function, then epigenetic modifications (changes of such as methylation, acetylation and ubiquitination that can cause changes in DNA packaging and , yet do not affect DNA sequence) must be responsible.

Centromere Proteins

In higher eukaryotes no specific centromere-nucleating sequence has been identified, which suggests the centromere is epigenetically regulated. One of the hallmarks of centromeric chromatin is the presence of CENtromere Protein (CENP) A.

CENP-A is a H3 variant exclusive to centromeres; CENP-A containing

are interspersed with those containing H3 (Blower, Sullivan et al. 2002).

CENP-A has been shown through a variety of studies to be necessary for centromere

function. Recent data suggests the protein complex Mis18 is, in turn, necessary for the

recruitment of CENP-A. Reduction of any of the Mis18 components results in

diminished CENP-A localization, accompanied by errors in chromosome alignment and

segregation (Fujita, Hayashi et al. 2007). The complete absence of CENP-A, as seen in

null mice, results in a high degree of chromosome missegregation and early embryonic

lethality (Howman, Fowler et al. 2000). Because of this functional dependence, it had

been hypothesized that CENP-A acts as a determinant of centromere location and

function. However, it has been shown by over-expression studies that all sites of CENP-

A localization do not become functional centromeres (Van Hooser, Ouspenski et al.

9 2001). Thus, while necessary, CENP-A is not sufficient for determining centromere location and no other obvious candidates have been identified. CENP-A is also necessary for the recruitment of other proteins that are involved in centromere function (reviewed in

Vos, Famulski et al. 2006) Not all the proteins associated with the centromere establish and maintain function; a large group of proteins form the kinetochore, the structure that mediates the interaction between the centromere and the spindle microtubules.

The kinetochore is the “work ” of the centromere. It is a trilaminar structure that performs at least three functions in the cell: 1) it facilitates the attachment of chromosomes to the spindle, 2) it mediates the subsequent movement of the chromosomes on the spindle and 3) it plays a role in control. There are two main “geographical” areas of the kinetochore: the inner centromere region and the outer kinetochore (Table 1.1; Figure 1.1).

The inner centromere region (ICR) is located between sister chromatids. Proteins known to be at the ICR are mitotic centromere-associated kinesin (MCAK), Shugoshin and the chromosomal passenger complex (CPC). The CPC is composed of the proteins

Aurora B, inner centromere protein (INCENP), and Dasra/Borealin. These proteins are involved primarily in sister chromatid cohesion, but also function in bundling and depolymerization (Vagnarelli and Earnshaw 2004; Vader,

Kauw et al. 2006; reviewed in Vos, Famulski et al. 2006; reviewed in Boyarchuk, Salic et al. 2007).

10 Table 1.1 Examples of proteins that localize to the centromere and kinetochore and their functions (Cleveland, Mao et al. 2003; Vagnarelli and Earnshaw 2004; Dawe and Henikoff 2006; reviewed in Vos, Famulski et al. 2006; Fujita, Hayashi et al. 2007).

Mammalian Protein Name Function Aurora B Phosphorylates and inhibits MCAK Bubs Mitotic checkpoint protein CENP-A variant, forms centromere-specific CENP-B Not essential; May aid in determining centromere location CENP-C “Kinetochore foundation protein,” recruits other essential proteins CENP-E Motor protein CENP-F Stabilizes kinetochore/microtubule interactions CENP-G May model chromatin for kinetochore function CENP-H “Kinetochore foundation protein,” recruits other essential proteins CENP-I Recruits other proteins such as CENP-F CENP-U Unknown CLIP-170 Motor protein Dasra/Borealin Facilitates interaction of Survivin and INCENP Motor protein INCENP Bundle microtubules Mads Mitotic checkpoint protein MCAK Depolymerizes microtubules Mis12 “Kinetochore foundation protein,” recruits other essential proteins Mis18 Recruits CENP-A Shugoshin Maintains sister chromatid cohesion Survivin Mediates localization of the CPC to the ICR

11

Figure 1.1 Regions of the centromere and kinetochore. Adapted from Cleveland et al. 2003.

12 The outer kinetochore is the main site of microtubule binding. Mis12, CENP-C

and CENP-H are known as “kinetochore-foundation” proteins. They are constitutively

present and recruit multiple transitory proteins. Also present at the outer kinetochore are

motor proteins (e.g., CENP E, CLIP-170, and dynein) that move chromosome along the

spindle and checkpoint proteins (such as the Mads and Bubs) that block a cell from

entering if a chromosome is not attached to the spindle (Table 1.1) (Cleveland,

Mao et al. 2003; Dawe and Henikoff 2006; Vos, Famulski et al. 2006).

What is A Good Strategy to Study the Centromere?

Studying centromeres and identifying the truly essential components is

challenging. As described in the studies of CENP-A, knocking-out even one component can result in lethality. One strategy to overcome this obstacle is studying naturally occurring centromeric variants—e.g., individual error-prone chromosomes in an otherwise normal, and viable, cell. The only endogenous human or mouse chromosome not necessary for cell and animal survival is the Y; i.e., the only known Y-linked genes are involved in testis development and (reviewed in Noordam and

Repping 2006), which, as evidenced by females, are not indispensable to an individual.

The Y chromosome therefore provides a unique reagent to study centromere structure and function.

What Is Known About the Y Centromere?

While little is known about most centromeres, the Y chromosome centromere remains the least well characterized. Although enigmatic, the mammalian Y centromere

13 is known to be a “minimalist” compared to all other centromeres. The human Y has been shown to have an approximate 20 fold reduction in the length of the higher order alphoid repeat structure by comparison with other chromosomes (Tyler-Smith and Brown 1987).

Not surprisingly, the amount of CENP-A that localizes to the human Y is also greatly reduced (Irvine, Amor et al. 2004). The mouse Y centromere appears to deviate even further from “normal,” as none of the known mouse satellite variants have been localized to the Y (Pardue and Gall 1970; Broccoli, Miller et al. 1990; Kuznetsova, Prusov et al.

2005). Further, both mouse and human Y chromosomes lack the centromeric protein

CENP-B, which is constitutively present at all other centromeres (Masumoto, Masukata et al. 1989; Broccoli, Miller et al. 1990; Earnshaw, Bernat et al. 1991)

It has been hypothesized that these differences make the Y centromere vulnerable to “attacks” such as or functional replacement by other sequences. For example, formation of a neocentromere has been suggested as particularly likely on the

Y chromosome, given the largely heterochromatic content of the arms (Warburton 2004).

Alternatively, the native Y centromere might be silenced by the acquisition of an active exogenous centromere. Indeed, the mouse Y* chromosome provides just such an example; an active centromere (of unknown origin) is located at the distal end of the q arm, while the original centromere, still located in the original position, is silent (Eicher,

Hale et al. 1991). Although such a specific defect or mutation has yet to be identified, work presented in Chapter 2 describes the structure and segregation behavior of an error- prone Y chromosome. This chromosome, along with other such variants, serves as an opportunity to study and elucidate the truly essential components of a functional

14 centromere. In turn, these studies can provide insight into the mechanisms of segregation errors, which have important implications for the genesis of human aneuploidy.

15 Environmental Toxins and

Few can argue that the “Chemical Revolution” that began in the mid 1900’s made

day-to-day activities more convenient and paved the way for modern living. Pesticides,

plastics and all-in-one cleaners are a few examples of the “essential” components of

today’s society. However, an important consequence of these advancements is the

chemical footprint they leave on the environment. There are over 80,000 chemicals

registered for commercial use in the United States (Newbold, Padilla-Banks et al. 2006)

and research suggests that at least some leach into the food and water supplies. In a

recent study, 167 different contaminants including pesticides, phthalates, and plastic

polymers were found in the blood and urine of nine adults. More alarming, an average of

200 contaminants was found in umbilical cord blood samples of newborn infants

(http://www.ewg.org/reports/bodyburden1/es.php). Thus, there is little doubt that

humans are being exposed to myriad compounds that can bioaccumulate; the question is

what are the consequences? Of the many possible repercussions that can be imagined, the effects of environmental toxins on fertility are of particular interest.

Is the Incidence of Human Infertility Increasing?

It has been suggested that human fertility is declining. Since the CDC began collecting data in 1996, the number of ART cycles begun each year has steadily increased; the number of cycles begun in 2004 was nearly twice that recorded in 1996

(CDC 2006). Certainly use of these technologies has increased as awareness and accessibility have risen, however, recent research suggests that infertility itself is

increasing. According to the National Survey of Family Growth published by the U.S.

16 National Center for Health Statistics, the number of women self-reporting problems

conceiving and/or maintaining a pregnancy rose from 4.9 million in 1988 to 6.1 million

in 1995 and 7.3 million in 2002. There has been a trend in women waiting until later in

life to start a family, which could affect fertility, yet, surprisingly, the most dramatic

increase in self-reported problems was among women under the age 25 (Chandra 2005).

There are many possible causes for such a decline in fertility, however, the following

section will focus only on potential environmental influences on human reproduction.

Additionally, there are several ways a person could be exposed to toxins; the studies

summarized here will involve three methods in particular: occupational exposure, routine

“day-to-day” exposure and medical (i.e., as part of treatment) exposure.

Impact of Environmental Toxins on Male Infertility

Medical Exposure

Frequently, medical therapies have negative side effects that must be endured for

the greater benefit of the treatment. Chemotherapy, often required to treat , can

have a lasting, and sometimes permanent, impact on fertility. Approximately 80% of

testicular cancer patients treated with either cisplatin or carboplatin returned to normal

sperm counts within five years of treatment (Howell and Shalet 2005). Similarly, full

recovery of spermatogenesis was seen in most patients treated with vinblastin, a drug

used to treat a variety of such as lung and testicular. Conversely, two common

treatments of Hodgkin’s lymphoma known as the MOPP regime (a combination of the

drugs procarbazine, chlormethine, vincristine and prenisolone) and ChIVPP

(chlorambucil, vincristine, procarbazine and prednisolone) have both been found to cause

17 permanent in men (Magelssen, Brydoy et al. 2006). Fortunately, options

such as pre-treatment semen cryopreservation are becoming more accessible for patients

concerned with post-treatment paternity.

Occupational Exposure

There are multiple examples in the literature of adverse reproductive effects in

men associated with occupational exposure to toxins. In one study, high levels of the

herbicides alachlor and atrazine, or the insecticide diazinon were significantly associated

with reduced semen quality (sperm number, morphology and motility) (Swan, Kruse et

al. 2003). Similarly, semen quality was also decreased in men with high levels of certain

phthalates, compounds used in the production of a wide range of products such as

plastics, solvents, pesticides and even perfumes and nail polish (Duty, Silva et al. 2003).

Men working as printers and exposed to the solvent toluene had significantly altered

levels of multiple hormones, including (Svensson, Nise et al. 1992). These

cases, and others like them (Table 1.2), provide evidence that occupational exposures can

have a profound impact on male fertility.

Day-to-Day Exposure

Not everyone has a job that requires the use of large quantities of chemicals,

raising the possibility that most people are safe from environmental toxins. However,

recent research would suggest that everyone, regardless of occupation, is subject to

exposure simply by living in the “modern” world. Some examples of compounds to which humans have widespread exposure are polychlorinated biphenyls (PCB’s),

18 polyhalogenated hydrocarbons and phthalates, all of which can alter endogenous levels, upsetting the androgen-estrogen balance. The use of estrogen as a growth promoter in farm animals has been suggested to pose a risk to the consumer, as residual hormones are present in meat (Sharpe 2003). Even some water supplies have been found to carry enough ethinyl estradiol to affect sex determination in fish (Jobling,

Beresford et al. 2002). Exposures to chemicals that alter endogenous hormone levels, in particular estrogen, and their potential impact on fertility have received much attention.

Specifically, the “estrogen hypothesis”, first suggested in 1993 (Sharpe and Skakkebaek

1993) and reasserted ten years later (Sharpe 2003) states that fetal exposure to increased levels of estrogen are to blame for an increase in testicular dysgenesis syndrome or TDS.

“Testicular dysgenesis syndrome” is an umbrella term that has come to encompass male fertility disorders that include cryptorchidism (undescended ), hypospadias (an abnormally placed urethral opening), low sperm count and testicular germ-cell cancer. Each of these is thought to have fetal origins, specifically from abnormal function of Sertoli and Leydig cells, the supporting somatic cells in the testis.

Sertoli cells are the first to differentiate in the developing testis, and support seminiferous cord formation, block cells from entering meiosis and stimulate Leydig cell function

(reviewed in Mackay 2000). In the adult testis, Sertoli cells physically and metabolically support spermatogenesis, and thus, fewer or abnormal Sertoli cells results in a reduction in sperm (Orth, Gunsalus et al. 1988). Leydig cells secrete hormones that are vital to downstream events such as descent of the testes (Hutson, Hasthorpe et al. 1997). The proliferation and development of Sertoli cells, and, in turn Leydig cells, is dependent on

19 the proper hormone signals in utero (Sharpe 2003). Disturbances in normal hormone

balances by endocrine disrupting chemicals would therefore have a lifelong impact.

Regardless of the cause, it has been questioned if, as the estrogen hypothesis asserts, there actually is an increase in TDS. There does seem to be a decline in sperm counts, however, much of this data is controversial and thus the subject of debate

(reviewed in Sharpe 2003). Arguments stem from the methods of collection; studies spanning decades are criticized as not having consistent counting methods, while many recent studies use men attending fertility clinics, which critics argue is a biased population. Whether there has been a change in the frequency of cryptorchidism is unclear, as there is not currently enough data to evaluate. New initiatives to study the frequency of cryptorchidism are underway though and hopefully will provide definitive evidence. It is clear, however, that the incidence of both hypospadias and testicular cancer are increasing, and taken together, TDS as a whole does seem to be on the rise

(reviewed in Sharpe, McKinnell et al. 2003).

20 Table 1.2 Examples of studies of the impact of chemical exposure on human fertility

Chemical Study Population Reference Male Cadmium Croatian men Decreased (Jurasovic, testis size Cvitkovic et al. 2004) Polychlorinated Prenatally Reduced (Guo, Hsu et al. biphenyls (PCBs) exposed sperm quality 2000) Taiwanese men Organophosphorus Mexican Abnormal (Sanchez-Pena, pesticides agricultural sperm DNA Reyes et al. workers condensation 2004) Mixed pesticides Italian Increased (Petrelli and greenhouse time to Figa- workers pregnancy Talamanca 2001) Female Dichloropiphenyl General Increased risk (Korrick, Chen trichloroethane (DDT) population of spontaneous et al. 2001) Chinese women abortion Polychlorinated American women Increased risk (Louis, Weiner biphenyls (PCBs) with a history of endometriosis et al. 2005) endometriosis Bisphenol A (BPA) Japanese women Increased risk (Sugiura- with a history of of Ogasawara, Ozaki et al. 2005) Cigarette smoke American fertility Reduced (Sharara, clinic patients follicular Beatse et al. reserve 1994)

21 Impact of Environmental Toxins on Female Infertility

Medical Exposure

Tragically, evidence of a once commonly prescribed compound actually having

an adverse effect on female fertility is readily available. Beginning in the late 1940’s and

continuing for approximately 30 years, physicians administered diethylstilbestrol (DES),

a synthetic estrogen, to help prevent miscarriages. Unfortunately, DES did not prevent miscarriages, and, in fact, actually had an adverse effect on the exposed fetuses.

Daughters of DES-exposed women have an increased risk of several reproductive

diseases, including vaginal clear cell adenocarcinoma (Herbst, Ulfelder et al. 1971),

breast cancer (Palmer, Hatch et al. 2002), reduced fertility (Senekjian, Potkul et al. 1988)

and abnormal pregnancies (Swan 1992). Adverse effects are not limited to daughters, but

have also been reported for sons of DES-exposed women, such as increased risks of

epididymal cysts and hypotrophic testes (Gill, Schumacher et al. 1979).

Occupational Exposure

Women are also at risk of occupational exposure to environmental toxins. In a

study of women who worked with agricultural pesticides, significantly more menstrual

cycle abnormalities (longer cycles, intermenstrual bleeding and missed periods) were

found than in women who did not have contact with pesticides (Farr, Cooper et al. 2004).

Female workers exposed to ethylene glycol ethers (EGE), solvents used in a variety of

industries such as the manufacturing of electronic components and photography, had a

significant increase in spontaneous abortions (Correa, Gray et al. 1996). Further,

although a mechanism has not yet been determined, routine exposure of dental assistants

22 to nitrous oxide has also been suggested to impair fertility (Rowland, Baird et al. 1992).

These are just a sampling of studies suggesting that, as in males, occupational exposures can have a negative impact on female fertility.

Day-to-Day Exposure

As seen with men, women are also exposed to toxins in their everyday lives.

Phthalates are found in products such as nail polish, hair spray and cosmetics. A study of

Indian women with a history of endometriosis found a significant correlation between the concentration of phthalates in blood samples and the severity of the disorder (Reddy,

Rozati et al. 2006). Even diet has been linked to infertility. Increased seafood consumption, concurrent with raised blood levels of mercury, was found to be associated with reduced fertility in both men and women in Hong Kong (Choy, Lam et al. 2002).

Other such alarming examples of such day-to-day exposures and their impacts on fertility are provided in Table 1.2.

Due to a recent, inadvertent exposure to a commercial cleanser in our animal facility, the rest of this section will focus on a family of everyday chemicals known as quaternary ammonium compounds. These compounds are found in countless household products such as cleaners and cosmetics (Table 1.3), yet the potential effects on fertility in either sex have not yet been thoroughly studied.

23 What are Quaternary Ammonium Compounds?

Quaternary ammonium compounds, or “quats”, are a family of disinfectants labeled as effective against bacteria and numerous viruses such as Avian flu and HIV-1.

First engineered in 1935, quats have evolved, becoming very convenient for use (e.g., no mixing of components, long shelf-life, no odor). They function by disrupting the cell membrane, denaturing cellular proteins and interfering with and metabolic reactions. Although these modes of action are known for bacterial and viral cells, like most other commercial compounds, similar detrimental effects on animal cells and tissues have not been thoroughly investigated.

Because of the ease of use, coupled with their efficacy, quats are among the most commonly used disinfectant in animal facilities (Ingraham, 2003). From the time disinfectants were created in the late 19th century, their use has exponentially increased, as have, presumably, their safety and efficacy. The first disinfectant, carbolic acid, was introduced in the operating room by Sir Joseph Lister and though effective at preventing infections, caused skin burns. Conversely, most modern day disinfectants are marketed as safe for application around both humans and animals.

Our laboratory moved from Case Western Reserve University to Washington

State University in 2005 and observed significant changes in our mouse breeding colony.

After methodically changing conditions in an attempt to mimic those at Case, we discovered it was the heavy use of a quaternary disinfectant that was impairing breeding.

Both specific pathogen free (SPF) and conventionally caged animals were housed in the

WSU facility, thus prompting the liberal use of the disinfectant to block cross-

24 contamination. Chapter 3 of this dissertation describes the negative impact of a quaternary disinfectant on the reproductive health of laboratory mice.

25 Table 1.3 Examples of common quaternary ammonium compounds and the types of products typically containing each one.

Quaternary Ammonium Synonym Product Cetyl trimethyl ammonium chloride Shampoo, hair color Didecyl dimethyl ammonium chloride household and industrial disinfectants Ecolyst herbicide Quaternium-15 lotion Quaternium-18 deodorant Quaternium-52 baby wipes

26 Research Aims

The overall objective of the studies described in this dissertation was to

investigate two factors that may negatively affect fertility: chromosome segregation

errors and environmental toxins. Two separate studies are described, one focusing on a

missegregation-prone Y chromosome and the other on the detrimental affects of a specific disinfectant on animal health.

The studies in Chapter 2 investigated the occurrence of hermaphrodites on the inbred mouse strain A/HeJ. The hermaphroditism trait was transferred concurrently with the YA/HeJ chromosome to the C57BL/6 background, thus indicating the trait was a

product of the YA/HeJ. Accordingly, the meiotic and mitotic segregation and structure of

the YA/HeJ chromosome were studied to further identify the source of .

The studies in Chapter 3 describe the effects of a quaternary ammonium

disinfectant on animal breeding. The ability to maintain a pregnancy, prolonged breeding

performance and pup nurturing both during a period in which the disinfectant was heavily

used and in a period following its removal from the animal facility are discussed.

The results and conclusions are discussed in Chapter 4 along with future

experiments.

27

Chapter 2: The mouse A/HeJ Y chromosome: Another good Y gone bad

Jodi M. Jackson1,2, Linda L. Washburn3, Laura Grindell1, Eva M. Eicher3, and Patricia A. Hunt1

1School of Molecular Biosciences, and Center for Reproductive Biology, Washington State University, Pullman, WA 99164

2Department of Genetics, Case Western Reserve University, Cleveland, OH 44106

3The Jackson Laboratory, Bar Harbor, ME 04609

Reference: Will be submitted

28 Abstract

In both humans and mice there have been numerous reports of Y chromosome abnormalities that interfere with sex determination. Recent studies in the mouse of one

such mutation have linked Y chromosome nondisjunction during preimplantation

development with disturbances in testis determination that result in hermaphroditism.

We report here that the mouse Y chromosome from the A/HeJ inbred strain induces similar aberrations in sex determination. Our analyses provide evidence, however, that the mechanism underlying these disturbances is not Y chromosome nondisjunction. On the basis of our findings, we postulate that a mutation at or near the centromere affects both the segregation and sex determining properties of the A/HeJ Y chromosome. This Y chromosome adds to the growing list of Y chromosome aberrations in humans and mice.

In both species the centromere of the Y is structurally and morphologically distinct from the centromeres of all other chromosomes. We conclude that these centromeric features make the Y chromosome extremely sensitive to minor structural alterations, and that our studies provide yet another example of a good Y chromosome gone “bad.”

29 INTRODUCTION

Mistakes in chromosome segregation play a significant role in the cause of human disease. For example, segregation errors during mitotic cell division are involved in the genesis and progression of a variety of human cancers (reviewed in Kops, Weaver et al.

2005), and mistakes during meiotic cell division are the leading cause of birth defects and miscarriages (reviewed in Hassold and Hunt 2001). Fundamental differences in the division process in meiotic and mitotic cells suggest that the underlying etiology of nondisjunction in the two types of cell division is different. Given that the centromere is a key player in chromosome segregation during both types of division, understanding centromere dynamics is fundamental to understanding the causes of human aneuploidy.

Although centromere function is highly conserved among organisms, centromere structure is subject to rapid evolution, and these structural changes play a role in (Zhao, Shetty et al. 2004). Little is known about variation in centromere sequence either within or among mammalian species. Nevertheless, it is clear from information about the mouse and human Y chromosomes that the Y centromere differs markedly from the centromeres of the other chromosomes in the . Cytogenetic banding techniques for visualizing centromeres demonstrate a single C-band negative chromosome – the Y chromosome - in both mouse and . In humans, the size of the Y centromeric alpha satellite array is approximately 5-10 fold smaller than the arrays present on the and , and the Y centromere is unique in lacking

CENP-B binding sites (Tyler-Smith and Brown 1987; Masumoto, Masukata et al. 1989).

Similarly, mouse X and autosomal centromeres contain four types of satellite sequences, but none have been detected on the Y, nor is CENP-B detectable at the Y centromere by

30 immunostaining (Pardue and Gall 1970; Broccoli, Miller et al. 1990; Kuznetsova, Prusov

et al. 2005). Thus, in humans and mice, the centromere of the Y chromosome is distinct.

Warburton (2004) has hypothesized that the mammalian Y centromere is “weak” and

thus easily mutated or inactivated.

The Y chromosome centromere of BALB/cWt mice provides an example of a

centromere that is prone to nondisjunction. In 1975 Whitten reported a high frequency of

hermaphroditism and a skewed sex ratio in BALB/cWt mice (Whitten 1975). Subsequent

studies demonstrated that BALB/cWt hermaphroditism was a property of the Y chromosome and the frequency of hermaphroditism was influenced by the maternal genetic background in F1 mice from matings of females of different inbred strains to

BALB/cWt males. The cause of BALB/cWt hermaphroditism was found to be due to nondisjunction of the BALB/cWt Y chromosome (YWt) such that this chromosome was

lost in a proportion of cells, resulting in XO/XY and XO/XY/XYY mosaicism (Beamer,

Whitten et al. 1978; Eicher, Beamer et al. 1980). Loss of the YWt chromosome during

development provides an obvious explanation for disturbances in sexual differentiation in

a proportion of XYWt animals. However, our view of the YWt chromosome as a simple

nondisjunction-prone chromosome was revised when we found that it is highly

nondisjunction-prone during early embryonic cleavage divisions, but exhibits normal

segregation behavior thereafter, with little if any increase in error frequency (Bean, Hunt

et al. 2001). These findings suggest that the first mitotic divisions during embryonic

development are subject to slight differences in cell cycle control that, in some way,

make the YWt chromosome vulnerable to centromere division errors. Furthermore, these

31 findings demonstrate that, under normal circumstances, cell cycle control mechanisms

can minimize the effects of a nondisjunction-prone chromosome.

Here we report a high frequency of hermaphroditism in A/HeJ strain mice. Given

the findings in BALB/cWt mice, we predicted that 1) the YA/HeJ chromosome would be

nondisjunction-prone, and 2) the defect in the YA/HeJ and YWt centromeres might be the

same, given that they share a common ancestor (i.e., Bagg Albino; Potter 1985). Our analysis, however, suggests that the YA/HeJ chromosome differs markedly from the YWt chromosome both structurally and phenotypically. Transfer of the YA/HeJ chromosome to

the C57BL/6J (B6) background demonstrates that the hermaphroditic phenotype in

XYA/HeJ mice is associated with the Y chromosome. Unlike the YWt chromosome, however, the high frequency of hermaphroditism in XYA/HeJ mice cannot be explained by

Y chromosome nondisjunction. We propose that a mutation occurred at or near the centromere of the YA/HeJ chromosome and this mutation 1) impairs centromere function,

resulting in a mild nondisjunction phenotype, and 2) introduces a position effect that

influences the expression of the Sry gene in a proportion of cells. We further suggest that

the mouse Y chromosome centromere is a centromere “on the edge.”

MATERIALS AND METHODS

Mice

Breeding stock of BALB/cWt, C57BL/6J and a C57BL/6J strain consomic for the

A/HeJ Y chromosome (C57BL/6J-YA/HeJ) were from the Eicher colony at The Jackson

Laboratory. All experiments were approved by the Institutional Animal Care and Use

Committees of The Jackson Laboratory, Case Western Reserve University, and

32 Washington State University. All three institutions are fully accredited by the American

Association for Accreditation of Laboratory Animal Care.

Southern Blot Analysis

For Southern analysis, DNA was prepared from tail clips and aliquots were

digested with Alu I (New England Biolabs, Beverly, MA) and electrophoresed through a

1% TAE gel run at 30 volts for approximately 36 hr. The DNA was transferred to Zeta-

Probe GT Genomic Tested Blotting Membrane (Bio-Rad Laboratories, Hercule, CA) by

blotting overnight in 0.4 N NaOH. The membrane was briefly washed in 2X SSC to

achieve a neutral pH and incubated in 0.1X SSC/0.5% SDS for 1 hr at 65ºC, followed by

four hrs in prehybridization buffer [4X SSCP (480mM NaCl, 60mM sodium citrate,

60mM Na2HPO4, 18mM NaH2PO4), 10X Denhardt’s solution (0.2% Ficoll, 0.2%

polyvinylpyrrolidone, 0.2% bovine serum albumin) and 1.0% SDS] at 65ºC.

Ready-To-Go DNA Labeling Beads (Amersham Biosciences, Piscataway, NJ) were used to label 250 ng of the probe, pErs5-532 (Eicher, Hale et al. 1991). The DNA

was denatured for 5 min at 100ºC, incubated on ice for 2 min and combined with the

labeling bead and 125 μCi of [α-32P]dCTP. The labeling reaction was allowed to proceed at 37ºC for 20 min and centrifuged through a Mini Quick Spin DNA Column (Roche-

Boerhinger Mannheim, Indianapolis, IN) to remove unincorporated nucleotides. The labeled probe (4.5-9.7 x 107 cpm) was added to 20 ml hybridization buffer (4X SSCP, 2X

Denhardt’s solution, and 1% SDS) and the membrane was incubated overnight at 65ºC.

After hybridization, one brief and nine 20 min washes were done in 4X SSC, 0.1% SDS

at 65ºC.

33 Differences in the intensity of pErs5-532 hybridization to DNA samples from

males and females make comparisons of bands difficult in a single exposure photo. Thus, for the sake of comparison, individual lanes were excised and the contrast was slightly

increased on female lanes and decreased on male lanes to make common bands

discernable.

Fetal Gonad Analysis and Chromosome Preparations

We transferred the A/HeJ Y chromosome to the C57BL/6J background by

repeated backcrossing of male progeny to C57BL/6 females. The resultant consomic

males (C57BL/6J-YA/HeJ) were mated to C57BL/6J females to generate fetuses for testis

differentiation and nondisjunction studies. Timed matings were used to obtain fetuses at

13.5 to 15.5 days of gestation. Fetuses were dissected from the uterine horns and staged

by limb morphology according to Theiler (1989). Individual of each fetus

were classified as an ovary, a testis, or an ovotestis (both ovarian and testicular tissue

present). Fetuses were genotyped by PCR for the presence of a Y chromosome using a

multiplex genotyping PCR assay on tissue lysate (Albrecht, Young et al. 2003). For

cytogenetic analysis, liver and fibroblast tissues were collected from all fetuses

containing testes or ovotestes. Air-dried chromosome preparations were made from the

liver, as described previously (Eicher and Washburn 1978). For the analysis of fibroblast

cells, primary cultures were established from limb and tail buds and chromosome

preparations were made from early passage cells, as described previously (Bean, Hunt et

al. 2001).

34 Fluorescent In Situ Hybridization Studies

Y Chromosome Mapping

A panel of nine probes was used to assess the structural integrity of the YA/HeJ chromosome. Figure 2.4 provides a schematic showing the expected site of hybridization on the Y and a brief description of each probe. Seven Y-specific probes were used:

Three BAC probes; b11, b63, and b72 (Mazeyrat, Saut et al. 1998); pErs5-532, a subclone of Ers5 (Epplen, McCarrey et al. 1982); Sry p422 (Gubbay, Collignon et al.

1990); pSx1 (Mitchell and Bishop 1992); and Y353/B (Bishop, Boursot et al. 1985). In addition, we used, pMov15/1, which hybridizes to the of the X and Y chromosomes and to sites on several autosomes (Harbers, Soriano et al. 1986), and pMSat5, which hybridizes to major satellite repeats of all chromosomes except the Y

(Varmuza, Prideaux et al. 1988). Probes were labeled according to the manufacturer’s instructions using either a digoxigenin- or biotin-nick translation mix (Roche-Boerhinger

Mannheim, Indianapolis, IN).

Labeled probes were denatured in Hybrisol VII (Oncor, Gaithersburg, MD) containing 10 μg mouse Cot-1 DNA (Invitrogen, Eugene, OR) for 8 min at 85ºC and incubated at 37ºC for 60-90 min. Preparations were soaked in 2X SSC for 5 min and denatured for 8 min at 85ºC before probes were added. After the addition of probe cocktails, preparations were hybridized overnight at 37ºC in a humid chamber.

Following hybridization, preparations were washed in 50% formamide/2X SSC at 39ºC for 5 min and 2X SSC at 39ºC for 5 min. Probes were detected with rhodamine-labeled anti-digoxigenin (Roche-Boerhinger Mannheim, Indianapolis, IN), FITC-labeled anti- digoxigenin (Roche-Boerhinger Mannheim, Indianapolis, IN), rhodamine avidin or FITC

35 avidin (Vector Laboratories, Burlingame, CA). DAPI (4’-6-Diamidino-2-phenylindole;

Sigma, St. Louis, MO) was used to counterstain the chromatin.

Aneuploidy Analysis

Prior to hybridization, fibroblast and liver preparations were dehydrated in an

ethanol series, air dried, denatured in 70% formamide/2X SSC at 65ºC for 2 min, quenched in ice-cold 70% ethanol for 4 min, dehydrated in an ethanol series, and air

dried. A mouse Y chromosome paint probe (Cambio-Open Biosystems, Huntsville, AL)

was denatured for 10 min at 65ºC and the labeled X chromosome probe, DXWas70, was denatured at 85ºC for 8 min. Following denaturation, both probes were incubated at 37ºC for 30-60 min, added to the denatured tissue preparations, and the preparation was hybridized overnight at 37ºC in a humid chamber. Following the hybridization, preparations were washed in 50% formamide/2X SSC at 39ºC for 5 min, in 2X SSC at

39ºC for 5 min, and were detected with rhodamine-labeled anti-digoxigenin (Roche-

Boerhinger Mannheim, Indianapolis, IN). Prior to analysis, preparations were counter stained with DAPI (4’-6-Diamidino-2-phenylindole; Sigma, St Louis, MO).

Meiotic Preparations

Diakinesis and Metaphase II

For the analysis of cells at diakinesis and metaphase II of meiosis, air dried

chromosome preparations were made from the testes of adult males using a slight

modification of the method developed by Evans (1964) as described previously (Cherry,

36 Hunt et al. 2004). Prior to analysis, chromosomes were stained with DAPI (4’-6-

Diamidino-2-phenylindole dihydrochloride; Sigma, St Louis, MO).

Pachytene

To analyze between the X and Y chromosomes, surface spread

pachytene preparations (Peters, Plug et al. 1997) were made and immunostained with

SCP3 antibody, as previously described (Cherry, Hunt et al. 2004). Preparations were hybridized with the pseudoautosomal probe, pMov15/1 using the hybridization

methodology described above. Following hybridization, pMov15/1 was detected with

FITC-labeled anti-digoxigenin (Roche-Boerhinger Mannheim, Indianapolis, IN) and the

DNA was counterstained with DAPI (4’-6-Diamidino-2-phenylindole; Sigma, St Louis,

MO).

RESULTS

Hermaphroditism is a feature of the A/HeJ strain

In 1993, an A/HeJ mouse with abnormal external genitalia was sent to the Eicher

laboratory for evaluation and determined to be a true with both ovarian

and testicular tissue. The skewed sex ratio in The Jackson Laboratory A/HeJ production

colony (currently 46.8% males among liveborn progeny as compared with 50.6% males

in the related A/J strain) and the presentation of this hermaphrodite prompted further investigation of sexual differentiation in the A/HeJ strain. Visual analysis of the gonads

of adult nonproductive males culled from the A/HeJ production stock between 1993 and

2005 revealed that ~4% were overt hermaphrodites. An additional 17% of nonproductive

37 males had abnormally small testes and no epididymal sperm. Although this suggests an

unusually high frequency of aberrant testis differentiation, it almost certainly

underestimates the frequency of hermaphroditism in these mice for three reasons. First,

if sufficient testicular tissue is present, hermaphrodites are fertile and would escape

detection in a screen of nonproductive males. Second, the external phenotype of

hermaphrodites varies depending upon the amount of testicular tissue present, and XY

animals with an external female phenotype – although nonproductive - would not be detected in this screen. Third, because A/HeJ is an albino strain, mammae pigment (a phenotypic feature specific to females) could not be used to ascertain hermaphrodites among individuals that would be sexed as males on the basis of external genitalia (Eicher,

Washburn et al. 1996).

Hermaphroditism is a property of the A/HeJ Y chromosome

To determine if the high frequency of hermaphroditism in A/HeJ mice was the result of a mutation on the Y chromosome, the X chromosome, or an , we examined the effect of the A/HeJ Y chromosome on the C57BL/6J background.

C57BL/6J-YA/HeJ consomic males were mated to C57BL/6J females to generate fetuses

for analysis. Fifty-three fetuses were genotyped by PCR for the presence of a Y

chromosome using a multiplex genotyping PCR assay on tissue lysate (Albrecht, Young

et al. 2003). Thirty had no detectable Y sequence and normal ovaries, thus were XX

females. Of the 23 Y-positive fetuses, one had a testis with attenuated cord growth

(Bouma GJ 2007, In Submission) and 5 (22%) had at least one ovotestis. These data

38 demonstrate that the hermaphroditic trait is associated with the YA/HeJ chromosome,

suggesting a Y chromosome mutation.

The YA/HeJ and YWt chromosomes are different on the sequence level

The BALB/c and A strains are descendents of Bagg Albino mice (Potter 1985).

Thus, it is possible that the YWt and YA/HeJ chromosomes are direct descendents of the

same ancestral Y chromosome and share a common defect. To assess sequence similarity

between these two Y chromosomes, we analyzed the Bkm repetitive sequences on the

short arm of the Y chromosome. In the absence of selective pressure, the GATA and

GACA repeats on the Y short arm are subject to rapid evolution. As shown in Figure 2.1, genomic DNA digested with AluI, and probed with the Bkm-detecting pErs5 probe

(Epplen, McCarrey et al. 1982) results in a complex banding pattern that allows for the differentiation of the Y chromosomes of mouse substrains. (Note, there are a few Bkm- related sequences present elsewhere in the genome, as evident by some hybridizing bands derived from XX DNA.) Comparison of the Y-specific bands demonstrates different patterns on the YWt and YA/HeJ chromosome (Figure 2.1) and stronger similarity between

YA/HeJ and the two other A strain Y chromosomes than with the BALB strain Y

chromosomes.

39

Figure 2.1 Southern analysis of Bkm repeats in genomic DNA Mouse genomic DNA (female and male) digested with AluI and probed with pErs5-532 (Epplen, McCarrey et al. 1982). Red brackets denote a region of the blot where the differences in Y-specific banding patterns between the A/HeJ and BALB/cWt Y chromosomes are most obvious.

40 The Y A/HeJ chromosome is prone to mitotic mis-segregation

The occurrence of hermaphrodites among the progeny of C57BL/6J-YA/HeJ males

suggests that aberrations in sexual differentiation in A/HeJ mice are a property of the Y

chromosome. This phenotype is reminiscent of the hermaphroditism in BALB/cWt mice

caused by Y chromosome nondisjunction during early embryonic cleavage divisions

(Bean, Hunt et al. 2001). For this reason, we tested the hypothesis that the YA/HeJ chromosome is nondisjunction-prone by analyzing the sex chromosome constitution in

C57BL/6J-YA/HeJ fetuses. Based on the results of studies of the BALB/cWt strain, we

expected that: 1) Mid-gestation fetuses carrying the YA/HeJ chromosome would exhibit a

high frequency of sex chromosome aneuploidy, 2) the level of aneuploidy would be most

pronounced in hermaphrodites, and 3) Y chromosome nondisjunction would be limited to

the early cleavage divisions, thus the frequency of aneuploidy in the two tissues analyzed

(liver and fibroblast cells) would be very similar (see Bean, Hunt et al. 2001). As a

control, we used C57BL/6J males because these mice have no increased incidence of

hermaphroditism.

Table 2.1 shows a comparison of Y chromosome aneuploidy in the three types of

Y-bearing fetuses (i.e., C57BL/6J males, C57BL/6J-YA/HeJ males and C57BL/6J-YA/HeJ hermaphrodites). Because artifactual chromosome loss is common in cytogenetic preparations, hyperploidy but not hypoploidy levels were used to obtain the most conservative estimate of Y chromosome nondisjunction. Although hyperploidy was higher in hermaphrodites, our results did not conform with the predictions based on

BALB/cWt males. First, gonadal phenotype did not predict hyperploidy levels.

Although the highest levels of hyperploidy were observed in hermaphrodites, the

41 Table 2.1 Analysis of Y chromosome hyperploidy and gaps

na Total Cells Hyperploidy (%) Gaps (%)b Liver C57BL/6J-YA/HeJY Hermaphrodite 7 700 14 (2.0) 336 (48.0) Male 7 695 9 (1.3) 460 (66.2) C57BL/6J Male 9 734 1 (0.1) 39 (5.3)

Fibroblasts C57BL/6J-YA/HeJY Hermaphrodite 7 678 27 (4.0) 50 (7.4) Male 7 654 17 (2.3) 22 (3.4) C57BL/6J Male 9 805 19 (2.4) 2 (0.2) a Indicates the number of individual fetuses bA gap was scored if there was an obvious space between sister centromeres

42 difference between A/HeJ males and hermaphrodites was not significant in either cell type. Second, although the level of Y chromosome hyperploidy was increased in

C57BL/6J-YA/HeJ hermaphrodites by comparison with controls, the difference reached significance in liver but not in fibroblast cells (χ2 =12.0, p<0.001, and χ2 =3.2, p=0.07, respectively). Third, the nondisjunction profiles in liver and fibroblast cells from the same individual were markedly different in hermaphrodites (Figure 2.2). These results are in striking contrast to BALB/cWt hermaphrodites, where nondisjunction limited to the first several mitotic divisions produces nearly identical patterns of mosaicism in different tissues (Bean, Hunt et al. 2001).

The YA/HeJ centromere exhibits morphological aberrations

In the course our analysis, we noted the frequent occurrence of a pronounced space between the sister centromeres of the YA/HeJ chromosome (Figure 2.3), which suggested that the nondisjunction phenotype of this chromosome resulted from premature separation of sister chromatids. Accordingly, we analyzed the metaphase morphology of the chromosome in greater detail. As shown in Table 2.1, the incidence of “gaps” (a space between sister centromeres) was highly dependent on cell type. The phenotype, however, was significantly increased in C57BL/6J-YA/HeJ animals (both males and hermaphrodites) by comparison with controls in both fibroblast (χ2=21.65, p<0.0001 and

χ2=55.24, p<0.001 for C57BL/6A/HeJY males and hermaphrodites, respectively) and liver cells (χ2=582.09, p<0.0001 and χ2=338.1, p<0.0001 for C57BL/6J-YA/HeJ males and hermaphrodites, respectively). Furthermore, in individual animals the frequency of

43 a

10 9 8 7 6 5 4 3

Y Hyperploidy (%) Y Hyperploidy 2 1 0 0 1020304050607080 Y Gaps (% )

b

10 9 8 7 6 5 4 3

Y Hyperploidy (%) 2 1 0 0 1020304050607080 Y Gaps (% )

Figure 2.2 The frequency of Y chromosome hyperploidy and of chromatid gaps are not correlated in individual C57BL/6J-YA/HeJY animals. Graphs provide frequency of hyperploid cells and Y chromosomes exhibiting a gap between sister centromeres in liver (squares) and liver (triangles) cells in Y-bearing (a) hermaphroditic and (b) male fetuses. Identically colored symbols represent the two tissues from an individual animal.

44

Figure 2.3 Representative images of morphological aberrations observed during analysis of the A/HeJ Y chromosome in liver and fibroblast cells. X and Y chromosomes were identified by FISH using a Y-specific FISH paint probe (green) and the X-specific probe, DXWas70 (red). DAPI stained images show: (a) a morphologically normal Y chromosome, (b) a Y with a gap between sister centromeres and, (c) a Y chromosome exhibiting premature separation of sister chromatids.

45 YA/HeJ chromosomes exhibiting gaps was not correlated with phenotype (i.e., male versus hermaphrodite) or with the level of Y chromosome hyperploidy (Figure 2.2).

The YA/HeJ chromosome segregates faithfully in meiosis

Both the increase in hyperploidy and the gap phenotype could be explained by a

structural aberration that affects centromere function. We reasoned that such an

abnormality might create problems during meiotic cell division. Extensive meiotic

analysis, however, revealed no aberrations. Analysis of 50 pachytene cells from

C57BL/6J-YA/HeJ males demonstrated normal synaptic behavior, with synapsis between

the X and YA/HeJ chromosomes limited to the small psuedoautosomal region (Table 2.2).

Analysis of cells at diakinesis/metaphase I revealed no increase in premature separation of the X and Y chromosomes, suggesting that crossing-over occurred and that a was sustained (Table 2.2). Lastly, the analysis of metaphase II cells revealed no difference in the percentage of X- versus Y-bearing cells (Table 2.2), nor was there evidence of gaps or premature separation of the Y chromatids in metaphase II cells (data not shown).

46 Table 2.2 Meiotic Analysis in C57BL/6J-YA/HeJY males

----- Pachytene ------Diakinesis ------Metaphase II --- XY XY Not XY XY Not X-bearing Y-bearing Paired Paired Paired Paired (%) (%)

C57BL/6J-YA/HeJY 55 1 106 5 52 (42) 73 (58)

C57BL/6J 43 0 113 4 67 (46) 78 (54)

47 The structural integrity of the YA/HeJ chromosome

To assess the structural integrity of the A/HeJ Y chromosome, we conducted

FISH analysis using a panel of Y chromosome-specific probes. A schematic indicating

the chromosomal localization on the mouse Y chromosome of the loci detected by these

probes is shown in Figure 2.4. We also examined the localization pattern of pMSat5, a

probe that hybridizes to the satellite repeats found at the centromere of all mouse

chromosomes except the Y chromosome. Thus, pMSat5 would hybridize to the Y

chromosome only if this Y had acquired centromere sequences from another

chromosome.

In addition to the YA/HeJ chromosome, we evaluated the nondisjunction-prone

BALB/cWt Y chromosome, which is subject to missegregation during the early

embryonic divisions (Bean, Hunt et al. 2001). As controls we included the Y

chromosomes from the BALB/cBy and C57BL/6J inbred strains, which do not exhibit

increased nondisjunction or elicit hermaphroditism. A sequential two-color FISH

analysis established the relative placement of each probe (e.g., Figure 2.5) and no

obvious structural abnormality was detected in either the YA/HeJ or YWt chromosome.

Indeed, the hybridization patterns were identical for all four Y chromosomes. Further,

pMSat5 hybridized strongly to all other chromosomes in the complement but was not detectable on any of the Y chromosomes examined.

48

Figure 2.4 Schematic showing the normal location and a brief description of the eight FISH probes used in the structural analysis of the A/HeJ Y chromosome. In addition, pMSat5, which detects major satellite repeat sequences at the centromeres of all chromosomes except the Y, was used to test for the acquisition of centromere sequences.

49

Figure 2.5 Structural analysis of the A/HeJ Y chromosome. Two examples of the sequential hybridization procedure employed (a-d) a Y chromosome (a) stained with DAPI, (b) hybridized with pMov15/1 (red signal), (c) hybridized with b63 (green signal) and, (d) merged image. (e-h) a Y chromosome (e) stained with DAPI, (f) hybridized with pErs5, (red signal), (g) hybridized with b72 (green signal) and, (h) merged image.

50 DISCUSSION

The skewed sex ratio and increased frequency of hermaphroditism in A/HeJ mice prompted us to examine the cause of the sexual aberrations these mice. To determine if a mutation on the Y chromosome is responsible, we transferred the YA/HeJ chromosome to the C57BL/6J background by repeated backcrossing to C57BL/6 females. We found that some XY mice on the resulting C57BL/6J-YA/HeJ consomic strain develop ovotestes, indicating that the aberrations in sexual differentiation in A/HeJ mice are a property of the Y chromosome.

Mice of the BALB/cWt inbred strain exhibit a similar sex ratio distortion and increase in hermaphroditism, a phenotype that segregates with the YWt chromosome. A proportion of bearing YWt develop as phenotypic females or hermaphrodites due to loss of the Y chromosome in cells of the differentiating XY gonad (Whitten 1975;

Beamer, Whitten et al. 1978; Eicher, Beamer et al. 1980). The errors that result in Y chromosome loss, however, are limited to a brief window of development, i.e., the first several cleavage divisions of the early embryo (Bean, Hunt et al. 2001). This implies that the first several mitotic divisions represent a transition from the error-prone female meiotic divisions to the stable, tightly controlled cell divisions that characterize fetal development. In the presence of a nondisjunction-prone chromosome (e.g., the

BALB/cWt Y chromosome), this can yield variable levels of aneuploidy in individual fetuses.

Given the hermaphroditism phenotype observed in both BALB/cWt and A/HeJ mice, we reasoned that, like the YWt , the YA/HeJ chromosome might be nondisjunction- prone, especially during the early cleavage divisions. Because the two strains share a

51 common ancestor, we considered that the defect in the YA/HeJ chromosome might be the

same defect as in the YWt chromosome. Our results strongly refute these conclusions,

providing structural evidence and segregation data that demonstrate that the YA/HeJ chromosome differs from the YWt chromosome and causes aberrations in sexual differentiation by an entirely different mechanism.

Structural evidence that the YA/HeJ and YWt chromosomes are different

The Bkm repeat sequences present on the short arm of the mouse Y chromosome

are subject to rapid evolution (Epplen, McCarrey et al. 1982) and can be used to

determine if specific Y chromosomes are derived from a common ancestral Y

chromosome (Eicher, unpublished). We found that the group of inbred strains analyzed

had characteristic Y-specific banding patterns (Figure 2.1), and that YWt and YA/HeJ

chromosomes were very dissimilar. Although these sequence differences do not

necessarily translate into functional differences between the YWt and YA/HeJ

chromosomes, they provide unambiguous evidence that these two Y chromosomes – and

likely their centromeres as well – are not identical. Further, we found that the Y-specific

band pattern for the YA/HeJ chromosome was highly similar to the other two A stains

analyzed. Given that a high incidence of nonproductive matings has not been reported in

other A strains, we think it likely that the mutation on the A/HeJ strain occurred after

A/HeJ and A were separated in the 1940s (Fox 1997).

52 Behavioral differences between the YA/HeJ and YWt chromosomes

Transfer of the YA/HeJ chromosome to the C57BL/6J inbred background clearly

demonstrates that the hermaphroditism phenotype is a property of the YA/HeJ chromosome. Our analysis of liver and fibroblast cells, however, provided evidence of only a very modest increase in Y chromosome nondisjunction compared to controls. In addition, we detected no aberrations in the synaptic or MI segregation behavior of the

YA/HeJ chromosome during meiotic cell division. Furthermore, the level of aneuploidy in

liver and fibroblast tissues from individual mice was not correlated with phenotype (i.e.,

whether the individual was a male or a hermaphrodite). The fact that the sex

chromosome mosaicism patterns in a given individual were strikingly different in the two

tissues analyzed also argues against a period of strong nondisjunction that is confined to

early cleavage divisions. Taken together, we can draw two conclusions. First, errors in Y

chromosome segregation alone do not provide a plausible explanation for the

disturbances in sexual differentiation induced by the YA/HeJ chromosome. Second, the

timing and mechanism of nondisjunction of the YA/HeJ chromosome are distinct from that

of the nondisjunction-prone YWt chromosome. This second conclusion is buttressed by

the unusual morphology of the YA/HeJ chromatids at mitotic metaphase, i.e., frequently a

pronounced space is evident between the sister centromeres of the YA/HeJ, but this is not

observed in the YWt chromosome.

Why does the YA/HeJ cause disturbances in sexual differentiation?

From our analyses, we conclude that the cause of hermaphroditism is different in

BALB/cWt and A/HeJ mice. Unlike the YWt chromosome, the YA/HeJ is only mildly

53 nondisjunction-prone, and there is no obvious correlation between the level of Y chromosome aneuploidy and hermaphroditism in XYA/HeJ mice. We suggest that the

YA/HeJ chromatid aberrations provide an important clue to the etiology of the hermaphroditism. That is, the propensity for mis-segregation and frequent appearance of spaces between YA/HeJ sister chromatids in metaphase cells likely results from a mutation

that alters and/or diminishes the centromere domain.

Physical mapping studies using a battery of Y-specific FISH probes did not detect

any structural aberrations on the YA/HeJ chromosome. However, because the physical

structure of the mouse Y centromere and pericentromeric region remain unknown, a

mutation in either of these regions might escape detection using this methodology.

Given the proximity of Sry to the Y centromere, an epigenetic change in the centromere domain could influence the expression of Sry as well as other adjacent genes. This is not without precedent: A of the Sx1 repeats on the mouse Y short arm was reported by Capel et al. to affect expression of Sry and lead to XY females (Capel, Rasberry et al.

1993). We hypothesize that the aberrations in testis differentiation in XYA/HeJ mice result

from a position effect that influences the expression of Sry and possibly other genes on the Y short arm in a proportion of cells. Further investigation is needed to determine if

Sry expression is decreased in the genital ridges of XYA/HeJ mice.

Our understanding of mammalian centromeres remains incomplete, but the

centromere of the Y chromosome is particularly enigmatic. In humans, centromeres are characterized by large blocks of alpha satellite repeats. By comparison with all other

chromosomes, the human Y has very low levels of these repetitive arrays (i.e., as low as

240kb on the Y vs. >4000kb on other chromosomes; Irvine, Amor et al. 2004). In

54 addition to sequence differences, the human Y centromere lacks the 17bp CENP-B box sequence motif that serves as a binding site for the centromere-specific protein, CENP-B.

The CENP-B protein is present at functional centromeres on all other chromosomes but is

not detectable at the centromere of the mouse or human Y chromosome (Masumoto,

Masukata et al. 1989; Broccoli, Miller et al. 1990; Earnshaw, Bernat et al. 1991).

Further, chromosome-specific analyses of the amount of CENP-A (a histone H3 variant

that defines the centromere-specific nucleosome) demonstrate very low levels on the

human Y (Irvine, Amor et al. 2004). Thus, data from several sources suggest that the Y

has a “minimalist” centromere. Although it remains unclear whether these differences

diminish the size of the kinetochore domain, the incidence of mitotic instability has been

suggested to be increased for the human Y chromosome by comparison with all other

chromosomes (Nath, Tucker et al. 1995).

Studies of aberrant centromeres in both human and mouse provide further

evidence that the Y contains a weak centromere (reviewed in Warburton 2004). Y

chromosomes that have been ‘pirated’ by a centromere from another chromosome have

been reported in both mice and humans (Eicher, Hale et al. 1991; reviewed in Warburton

2004). This aberration requires both the introduction of a new centromere and silencing

of the endogenous Y centromere. The idea that the Y has a weak centromere is further

enforced by the finding that the human Y appears to be a favored site of neocentromere

formation (Warburton 2004). Taken together, these data suggest that, by comparison

with the centromeres of other chromosomes, the Y centromere is comparatively small

and easily inactivated.

55 The mammalian sex chromosomes are thought to have evolved from a common autosomal chromosome (Ohno 1967). This, however, does not provide an explanation for the striking structural differences between the centromeres of the X and Y chromosomes (e.g., in humans the centromeric alpha satellite array on the X is approximately 2.2 -3.7 Mb while the Y array is only several hundred kb; Irvine, Amor et al. 2004) or for the comparatively weak of the Y centromere. Graves has postulated that the mammalian Y chromosome is slowly devolving and will ultimately disappear (Graves 2006). We favor a slightly different hypothesis: Unlike the X chromosome, which contains a host of essential housekeeping genes, the only essential functions of the Y chromosome are in the initiation of testis differentiation and spermatogenesis. The fact that the Y chromosome can be lost from cells without impacting their survival apparently results in a relatively low selective pressure on the centromere of the Y chromosome. In both mice and humans this has resulted in a minimalist centromere that cannot easily sustain mutations in centromere structure that reduce the size of the region. Intriguingly, a previous study attempted to determine if the length of the alpha satellite array on the human Y chromosome was correlated with the frequency of sex chromosome disomy in sperm from normal men and XYY males

(Abruzzo, Griffin et al. 1996). Although no significant differences were detected, the authors concluded that the design of their study may not have provided access to the most extreme size variants necessary to detect segregation effects. Indeed, based on our studies in BALB/cWt and A/HeJ mice, it is likely that individuals with high levels of sex chromosome mosaicism, sexual ambiguity, or infertility may provide the best population for detecting analogous Y centromere defects in humans. On the basis of the data from

56 humans and mice, we postulate that the nature of its centromere places the Y chromosome on the edge of dysfunction, and that the Y chromosome from the A/HeJ strain provides yet another example of a way in which this chromosome can “go bad.”

57 ACKNOWLEDGMENTS

This manuscript is dedicated to the memory of Barbara K. Lee, whose technical expertise

lives on in the hands of the trainees who were privileged to receive her tutelage and in

their trainees whose results are still compared to hers. We gratefully acknowledge Terry

J. Hassold for constructive comments on the manuscript. These studies were supported by NIH grants HD31866 and HD37502 (PAH) and GM20919 and RR01183 (EME).

58 Chapter 3: Disinfectants and Dysfunction: Solving the Mystery

Jodi M. Jackson1,2, Jodi Griswold2, Rhonda Skaggs3, Ailene Muhlhauser2, William Siems3 and Patricia A. Hunt2

1Department of Genetics, Case Western Reserve University, Cleveland, OH 44106

2School of Molecular Biosciences, and Center for Reproductive Biology, Washington State University, Pullman, WA 99164

3LBB2 Analytical Services, Department of Chemistry, Washington State University, Pullman WA 99164

Reference: Will be submitted

59

Abstract

Our laboratory relocated from Case Western Reserve University in Cleveland,

Ohio, to Washington State University in Pullman, Washington, and experienced a dramatic decline in the reproductive performance of our mouse colony. Pregnancy rates and survival to weaning were significantly decreased, while developmental defects, late fetal death and dystocia were increased. We also observed decreased egg and embryo quality, as well as accelerated transport through the reproductive tract. Environmental variables were changed to mimic conditions at Case, with little improvements in breeding performance. Through the analysis of chemical residue present on “clean” cages and increased awareness of vivarium personnel training and daily sanitation protocols, we identified the liberal use of a disinfectant that contained quaternary ammonium compounds as the source of the reproductive maladies. By eliminating the quaternary ammonium-containing disinfectant from the facility and changing the cage wash protocol, we were able to significantly improve the breeding performance of our mouse colony. Our experience suggests that quaternary ammonium compounds have a negative impact on animal reproduction. We suggest that animal exposure to chemicals should be limited as much as possible, as residue can adhere to caging materials and may not be removed by the wash process. Also, well-trained and observant animal care staff are invaluable to the success of a research program.

60

Introduction

The efficiency of breeding colonies can be influenced profoundly by

environmental factors, and most animal facilities go to great lengths to control

environmental variation by careful daily monitoring of temperature, humidity, lighting

and housing conditions. Moving breeders from one facility to another invariably impacts

breeding, and the experienced colony manager will anticipate a stress-induced decline in

reproductive performance followed by a spontaneous rebound after a brief period of

acclimatization. In the absence of a spontaneous rebound, the cause of reproductive

decline may be an environmental variable not present in the previous facility, and

experimenting with small changes (e.g. humidity levels, temperature, cage design, or

husbandry protocols) or carefully duplicating the previously successful breeding

environment is usually sufficient to restore reproductive performance. Occasionally,

however, the task of optimizing breeding is considerably more difficult because the

problem is due to a combination of stressors or because the source of the disturbance is

unexpected. We provide here an account of our efforts to resolve the detrimental effects

on a mouse breeding colony that became apparent after our laboratory relocated from

Case Western Reserve University in Cleveland, OH to Washington State University in

Pullman, WA. After months of carefully adjusting and monitoring all obvious variables, we reached the surprising conclusion that the problems in our mouse colony were related

to “modernized husbandry,” that is, automated cage washing and the liberal use in the

facility of disinfectants containing quaternary ammonium compounds.

61 Methods and Materials

Breeding Protocols

In our colony, inbred stains are maintained by trio matings (two females paired with one male) of sibling animals. Breeder cages are established when animals are 6-8 weeks of age and females are retired at or before 8 months of age. To obtain timed pregnancies, females are placed with males, checked daily for the presence of a plug, and separated from the male on the morning that a plug is found.

Superovulation

Young (4-6 week old) C57BL/6J females were stimulated via intraperitoneal injection of 5 I.U. Pregnant Mare Serum Gonadotropin (PMSG; Sigma, St. Louis, MO) followed 46-48 hours later by 2.5 I.U. human Chorionic Gonadotropin (hCG; Sigma, St.

Louis, MO). Following hCG injection, each female was placed with a male and checked the following morning for the presence of a copulation plug. Four to eight cell embryos were collected ~65 hours after hCG administration.

Methanol Cage Extractions

To obtain extracts for analysis, 5 cages were washed with a small volume of methanol using the following procedure: Approximately 40 mL of methanol (JT Baker,

Phillipsburg, NJ) was used to slowly and completely rinse the inside walls of one polysulfone microisolator cage. The methanol was transferred to a second cage, the rinsing procedure was repeated, and the methanol was transferred to the third cage. This procedure was repeated for two additional cages, thus combining the chemical residue

62 from five cages into one methanol sample. The resultant samples were collected and stored in glass vials that had been previously tested and demonstrated to be free of exogenous chemical residue.

Analytical Methods

Mass spectrometric analyses were performed using an Applied Biosystems/MDS

Sciex API 4000 LC/MS/MS System equipped with a heated TurboIon Spray source operated in positive-ion mode, and an Agilent 1100 Series HPLC system. Instrument control, data acquisition, and initial data analysis were performed by Analyst 1.3.1 software. The active ingredients in the quaternary ammonium disinfectant are dimethyl didecyl ammonium chloride (~4.352%) and n-alkyl dimethyl benzyl ammonium chlorides (4.095%); only the dimethyl didecyl ammonium (DDA) component was quantitatively analyzed. Dimethyl didecyl ammonium bromide (98%, Aldrich, St. Louis

MO) was used as a standard, with five standards ranging from 3.6 nM to 181 nM analyzed with each sample batch. Chromatographic separation was carried out using a

MM-5-C4W-1000 column (Micro-Tech Scientific Inc., Vista, CA). Nitrogen was used as the curtain gas (10 psi) and collision gas, and purified air was used as the source gas. The declustering potential was set at 30 V and the collision energy at 40V. Quantitative analyses of DDA were monitored as selected ion reaction m/z pair 326/186. Mobile phase A was 0.1% acetic acid (J.T. Baker, Phillipsburg NJ), and mobile phase B was acetonitrile (EMD Chemicals Inc., Gibbstown NJ). The column was equilibrated with the initial mobile phase composition (70% A) for 15 min before injection. The gradient was as follows: isocratic (70% A) for 5 min, followed by a linear decrease to 37.1% A at

63 16.5 min; 2% A at 17.5 min; 2% A at 27.5 min; 70% A at 30 min. The injection volume of all standards and samples was 5 uL, and the flow rate was 125 uL min-1.

Results

Problems in a new breeding colony are immediately apparent

During a 3 month period from January to March, 2005, we moved breeding stock from our colony at Case Western Reserve University (Case) to Washington State

University (WSU). The WSU vivarium houses pathogen-free mice as well as mice and rats that harbor pathogens. We moved breeding stock of four inbred strains and thirteen mutant lines and, within several months, all stocks were breeding. One of the first experimental studies that we initiated required large numbers of timed pregnancies. For these studies, C57BL/6J inbred strain females ordered from The Jackson Laboratory were placed with males at 6 weeks of age. Females were checked daily for the presence of a copulation plug and separated from males on the morning that a plug was found.

Although the mating behavior of our animals appeared normal, few mated females proved pregnant when examined 12 days after mating; for the first 4 months (April –

July), the pregnancy rate was 5/46 (10.9%). Based on our prior breeding experience with the strain and data from The Jackson Laboratory, these numbers are significantly below the 70% pregnancy rate expected for this strain (t=79.6, p<0.0001). Further, among females that became pregnant and were used to obtain late gestation fetuses, a variety of defects were observed among the offspring, including late fetal death, litters whose developmental stage did not correspond to their gestational age (i.e., either significantly delayed or accelerated in their development), and a variety of developmental defects

64 (e.g., one pup lacked eyelids, another had a perfectly formed head and upper torso, but lacked a lower torso and hind legs; Figure 3.1).

Upon closer examination, it became obvious that there were also problems in our breeder cages. We noted the loss of a number of pregnant breeder females to dystocia.

This apparent increase in delivery complications, coupled with the finding of late fetal demise in our studies of late gestation fetuses suggested that we were experiencing an increase in late fetal loss. In addition, both the survival and growth of newborn pups was poor. In some breeding trios as many as 50% of offspring were being lost prior to weaning and, even in litters without appreciable loss, the small size of pups precluded weaning on schedule. A comparison of productivity in C57BL/6J breeders in the WSU colony during the summer of 2005 and in the Case colony the previous summer confirmed our suspicions that breeding was significantly impaired: In the first 4 months after pairing, 112 weaned offspring were generated from 3 breeding trios at Case, whereas only 51 total offspring were produced at WSU from the same number of breeders in the same timeframe (t=4.3, p=0.013).

Ruling out the obvious candidates

To improve reproductive performance, we began to systematically investigate and modify environmental conditions. We started with the most obvious factors; diet, light and temperature, all of which play a significant role in mouse breeding performance

(Murray and Parker 2005). Mouse chow was changed to the diet used at Case, Purina

5010; the daily light/dark cycle was changed from 12on/12off to 14on/10off

65

Figure 3.1 A morphologically abnormal 18.5 day A/J fetus. The head and upper limbs have formed normally, but the mid-torso and lower limbs are completely absent.

66 (http://jaxmice.jax.org/library/faq/index.html); the ambient temperature was raised from

an average range of 68-70ºF to 70-72ºF. Further, we ruled out disrupting ultrasonic

noises using bat detection equipment and, to minimize potentially disturbing vibrations,

we moved the ventilation blower from the top of the cage rack to a wall mount. Lastly, in

early August, 2005, a dedicated vivarium staff member was assigned to our room and we

instituted a policy of limited access (e.g., once per/day for cage checks and/or changes).

To assess the effects of these minor environmental modifications, we utilized the simplest

variable, the plug to pregnancy rate in young (6-8 week old) virgin females. As shown in

Figure 3.2, collectively these changes (May-November, 2005) had a positive effect on

C57BL/6J productivity. Given the improved pregnancy rate at the end of 2005, we

concluded that stress (i.e., frequent disturbances and minor environmental factors) was

responsible for the initial poor reproductive performance in our WSU colony (Reeb-

Whitaker, Paigen et al. 2001; Peters, Bywater et al. 2002).

New research studies reveal continued reproductive disturbances

Confident that our reproductive problems were resolved, we initiated a new set of research studies that necessitated the use of exogenous hormones to stimulate the ovaries

of young (4-6 week old) C57BL/6J females. This technique is routinely used in our

laboratory to increase the number of eggs that can be obtained per female. Our

experience at Case led us to expect to 20-30 oocytes/female, but we found that animals

housed at WSU showed little or no response to the injected hormones. After several

attempts with hormones from different vendors, we concluded that the problem was our females, not a faulty batch of hormones.

67

Figure 3.2 Pregnancy rate in young (6-8 week) virgin C57BL/6J females. Females were paired with males, checked daily for the presence of a copulation plug, separated from males on the morning that a plug was detected (day 0), and checked for pregnancy on day 12. Bars denote the percentage of matings that resulted in pregnancies for each month; black arrows denote significant husbandry changes; red arrows indicate the timing of pinworm outbreaks in the vivarium.

68 The lack of response to exogenous hormones suggested defects in oocyte growth.

To further investigate the problem, we initiated studies of early cleavage embryos so that we could simultaneously assess egg quality (i.e., by comparing the proportion of early cleavage embryos and of eggs that remained unfertilized) and embryo development (i.e., by determining if cellular divisions occurred on schedule). Following fertilization, embryos undergo several cell divisions as they move through the reproductive tract. In the , embryos reach the uterus and initiate implantation 3.5 days after fertilization. As in our initial studies, females were stimulated with exogenous hormones.

Following hCG injection females were placed with males, checked the following morning for copulation plugs, and embryos recovered from the reproductive tract two days after mating. As in initial stimulation studies, the number of embryos recovered was low; 104 embryos/unfertilized eggs were recovered from 7 females (an average of 13 eggs ovulated per female), however, the vast majority were unfertilized eggs, and only 33

(31.7%) cleavage stage embryos were recovered. Although the majority of recovered embryos were at the appropriate developmental stage, (26/33 or 78.8% were at the 4 to 8- cell stage), surprisingly many of the embryos were recovered from the uterus. At two days post fertilization, embryos should still be in the fallopian tubes, with entry into the uterus occurring on day 3. Thus, finding embryos already in the uterus suggested accelerated transport through the reproductive tract. In short, our studies of eggs and embryos from females stimulated with exogenous hormones revealed three defects; 1) a poor response to exogenous hormones, 2) impaired oocyte quality, resulting in fertilization failure, and 3) abnormally rapid transport of fertilized embryos through the reproductive tract.

69 In the course of a literature search to understand the reason for the accelerated

transport of embryos to the uterus, we encountered a citation reporting altered embryo

transport as a result of exposure to the pesticide, methoxychlor (Cummings and Perreault

1990). Three of the five investigators housing animals in the WSU vivarium were

conducting reproductive toxicology studies using a variety of chemical compounds,

including methoxychlor. Thus, we began to suspect that the reproductive abnormalities in our animals were the result of inadvertent environmental contamination.

Are we victims of our own research?

To test the hypothesis that environmental contamination from chemicals used in research studies was negatively impacting our animals, we initiated studies to detect chemical contaminants. Using methanol, an effective solvent for organic compounds, we rinsed the inside walls of microisolator cages and analyzed the resultant extract via gas chromatograph-mass spectrometry (GC-MS). This technique allows for the detection and potential identification of individual chemicals in a complex mixture. Analysis of the cage extracts revealed a wide array of chemical compounds, even on cages extracted immediately after washing. After conducting experiments to account for contaminants from food and bedding materials, a number of unidentified compounds remained.

Surprisingly, however, none matched the profiles of the suspected chemicals, i.e., those currently being used in toxicology studies. After several months of refining the analysis

(e.g., comparing extracts from new cages before and after washing), we made two conclusions: First, the cage washer was not adequately cleaning caging materials and

70 second, a common chemical signature in all experiments was a component of the

quaternary ammonium disinfectant used in the facility.

Quaternary ammonium compounds, or quats, are effective disinfectants and are

commonly used in animal facilities, especially those maintaining pathogen-free animals

(Ingraham and Fleischer 2003). Both specific pathogen-free (SPF) and conventionally caged animals were being maintained in our facility, and a disinfectant containing two quaternary ammonium compounds (n-alkyl dimethyl benzyl ammonium chlorides and didecyl dimethyl ammonium chloride) was being used. The detection of these compounds in new cages following a single pass through the cage washer made us suspect that exposure to these quaternary ammonium compounds was the source of our breeding problems. Retrospective analysis of the breeding performance of our animals revealed two striking correlations that supported this hypothesis: First, the dedicated staff member assigned to our colony when we limited access in August of 2005 was wary of the use of chemicals in close proximity to animals. Thus, she was sparing in her use of the proscribed disinfectant. As seen in Figure 3.2, the first significant increase in pregnancy rates occurred during her first month as caretaker of the colony. Second, in mid-October, 2005 and mid-January, 2006 pinworms were detected in non-SPF animals in the facility. Infected colonies were treated with febendazole feed and, to contain the outbreak, the quaternary disinfectant was used to fog infected rooms and to wash floors and surfaces in all areas of the facility. A noticeable drop in the pregnancy rate in our colony was observed after each outbreak (Figure 3.2).

71 Discontinued use of the disinfectant but persistence of the chemical compounds

The presence of chemical residues in washed cages and the consistent finding of

contaminants from the quaternary disinfectant suggested that our animals were being

inadvertently exposed to chemicals. Accordingly, we made major procedural changes.

To address the concern that the cage washer was not adequately washing caging materials, we amended the washing protocol in February, 2006: We discontinued the

practice of recycling the final rinse water as a pre-wash rinse for the subsequent wash

load, added an additional wash cycle, increased the amount of detergent, and extended

the time of the final rinse. In March, 2006, we eliminated the quaternary ammonium-

containing disinfectant from the facility and replaced it with a disinfectant containing

sodium chlorite. Further, for more accuracy in monitoring the levels of quaternary

ammonium compounds, we switched to the use of liquid chromatography-tandem mass

spectrometry (LC/MS/MS) for the analysis of cage extract samples because it allows for

detection and quantification of a specific ion.

To monitor the persistence of the quaternary compounds in the environment, we

monitored caging materials for 8 months after the removal of the disinfectant from the

facility. As shown in Figure 3.3, the amount of quaternary ammonium residue detectable

by LC/MS/MS declined rapidly in the two months after the disinfectant was removed.

Nevertheless, detectable contamination persisted in the environment for many months.

72 180

160

140

120

100

80

concentration (nM) 60

40

20

0 January April May June August October November

Figure 3.3 Cage extracts reveal contamination with quaternary ammonium compounds. Random samples of microisolator cages in general circulation were taken at regular intervals during 2006. Each bar represents the concentration of didecyl dimethyl ammonium chloride detected in a methanol extract from 5 cages sampled after cage washing.

73 Inadvertent recontamination

To directly test the effect of quaternary ammonium compounds on rodent reproduction, we initiated controlled experiments in August, 2006. This was 5 months after the removal of the quaternary ammonium-containing disinfectant; at this time, pregnancy rates had remained above 50% for 5 months (Figure 3.2) and monitoring of cages indicated a significant reduction in residual quaternary ammonium contaminants

(Figure 3.3). To mimic the original exposure, a small group of breeder cages was exposed to aerosolized disinfectant 3 times/week. Precautions were taken to prevent contamination: First, the animals for these studies were housed in a separate room in the facility and aerosolized exposures were conducted in a designated transfer station; second, cages housing animals used in the study were rinsed in methanol to decontaminate them prior to washing and were washed and autoclaved separately from other cages and third, to remove contaminated water from the cage washer, a purge cycle was run after exposed cages were washed. Despite these precautions, ~3 months after controlled studies were initiated, we and another investigator housing SPF animals in the facility noted a resurgence of breeding problems in our colonies. We suspected that our attempt to conduct controlled experiments of animals exposed to quaternary disinfectant was re-contaminating the facility, and we immediately suspended the studies.

To determine the extent of contamination, we analyzed extracts from new cages after they were run through a wash cycle. As shown in Figure 3.4 (Load 1), the results of this analysis demonstrated levels of quaternary compounds similar to those observed one month after the disinfectant was removed from the facility. In addition, we found even higher levels (Figure 3.4, Load 2) in cages washed immediately after a load of

74 contaminated cages (i.e., those used in controlled exposure studies but “decontaminated” with a methanol rinse before washing). These results suggest that, regardless of the changes in the washing protocol - including the addition of a purge cycle after every load of contaminated cages – cages throughout the facility were being contaminated again with quaternary compounds. In a subsequent experiment, we compared extracts from cages before and after autoclaving in the presence of contaminated cages (data not shown). The levels of quaternary compounds were not increased in autoclaved cages, hence we concluded that the major route of contamination was via the cage washer.

75 140

120

100

80

60 concentration (nM) concentration 40

20

0 Load 1 Load 2 Load 3

Figure 3.4 Quaternary ammonium contaminants are transferred during cage washing. Each bar represents the concentration of didecyl dimethyl ammonium chloride present in a methanol extraction from five new, unused cages after a wash cycle. Didecyl dimethyl ammonium chloride was not detectable in extracts of the same cages prior to washing (data not shown). In load 1, new cages were washed alone. Following this load, ~60 dirty cages used in controlled exposure studies were washed. This load was followed by a purge cycle and, in the final two loads (Load 2 and 3), 5 new, unused cages were washed alone.

76 Discussion

We report here the results of our attempts to understand and improve the breeding

performance of our mouse colony following relocation to a new facility. Based on the

data presented here, we conclude that build-up of quaternary ammonium compounds in

the environment as a result of the heavy use of a common disinfectant was responsible

for the reproductive abnormalities we observed in our mouse breeding colony during our

first year at WSU. Specifically, pregnancy rates declined during times of increased

disinfectant use (i.e., immediately after pinworm outbreaks) and, after the disinfectant

was removed from the facility, we noted improved breeding performance, increased pup

survival, and an increase in the weight of weaning age animals.

Initially we believed that our problems were due to the over zealous use of a

quaternary ammonium-containing disinfectant in an attempt to maintain pathogen-free

status in a facility housing animals with a variety of pathogens. However, two of our

findings raise more generalized concerns about the potential effects of quaternary

ammonium compounds on human and animal health. Firstly, monitoring of cages for 8

months after the disinfectant was removed from the facility demonstrated that these

compounds persist in the environment. Secondly, our attempt to directly demonstrate that exposure impacts reproduction by running controlled exposure experiments on a very

small number of cages apparently resulted in re-contamination of the facility. These

findings, coupled with the wide range of reproductive effects observed in our animals

raise concerns about the safety of quaternary ammonium compounds, and further studies

of the effects of these chemicals on animal reproduction are clearly warranted. The

we observed during the peak exposure periods suggest that ovarian, uterine,

77 and mammary tissue function were all impaired. Given these widespread reproductive effects, we think it likely that one or both of the quaternary ammonium compounds present in the disinfectant exert their effect by influencing the brain. Studies to assess the endocrine profiles of exposed females are currently in progress.

The growing demand for pathogen-free animals requires reliance on chemical disinfectants. Our experience demonstrates, however, that making users aware of the fact that these chemicals are pesticides is critical. Quaternary ammonium compounds were first engineered in 1935, but successive generations of more stable compounds have been generated over the past 50 years (Rutala 1996). These compounds function by disrupting the cell membrane, denaturing cellular proteins and interfering with enzymes and metabolic reactions (Ingraham and Fleischer 2003), and they are effective against a wide variety of bacteria and viruses, including Avian flu and HIV-1. The ease of use of these chemicals (e.g., they are odorless, not corrosive to equipment, have a long shelf- life, are not affected by pH, and do not require extensive mixing of components) has made quaternary ammonium compounds popular disinfectants. However, our studies raise concern that, in addition to the intended bacterial and viral cell targets, animal cells may be equally vulnerable to the actions of these chemicals.

In addition to raising concern about the safety of quaternary ammonium containing disinfectants, our experience provides several additional lessons. First, as in countless other facilities, we rely on an automated cage washer and subsequent autoclaving to protect our animals. Many compounds, however, adhere to plastic and, as our experience demonstrates, chemical residue can be transferred to new cages during the washing procedure. By making simple modifications to the wash protocol we were able

78 to significantly reduce—although not eliminate—the amount of chemical build-up on cages. Thus, our experience demonstrates that limiting the number of chemicals used and minimizing direct exposure to animals and caging materials is critical. Given that

disinfectants are essential in controlling pathogens in a facility housing SPF animals, our

findings underscore the importance of proper training and monitoring. Lastly, and most

importantly, our experience demonstrates that well trained personnel who pay attention to

details and are able to detect subtle changes in animal behavior and health are invaluable

to researchers. Attention to seemingly insignificant details and meticulous record-

keeping allowed us to resolve the issues in our breeding colony and, quite simply, this would not have been possible without the help and cooperation of the animal care staff.

79 Chapter 4: Summary and Future Directions

Summary

This dissertation had two primary foci: chromosome segregation errors and environmental endocrine disruptors. Both can have widespread, lasting impacts on the fertility of the general population. Aneuploidy affects 10-30% of human conceptions and is the leading genetic cause of pregnancy loss and mental retardation (Hassold and Hunt

2001). Chromosome segregation errors are widely acknowledged as problematic and have been long-studied. The effect of environmental toxins on fertility, however, is a more recently recognized phenomenon that, while not historically appreciated, is receiving exponentially more attention. These two areas have been considered separately in this dissertation

The Mouse A/HeJ Y Chromosome: Another Good Y Gone Bad

The A/HeJ Y Chromosome Induces Hermaphroditism

The identification of an A/HeJ mouse with abnormal external genitalia prompted

an investigation of the occurrence of hermaphroditism on the strain at The Jackson

Laboratory (TJL). The sex ratio in the TJL A/HeJ production colony was found to be

skewed, with males accounting for only 46.8% of liveborn progeny. Further, adult

nonproductive breeders were examined and ~4% were found to be overt hermaphrodites

and an additional 17% had abnormally small testes and no epididymal sperm.

The observed hermaphroditism could be the result of a mutation on the Y

chromosome, the X chromosome or an autosome. C57BL/6J animals consomic for the

A/HeJ Y chromosome (C57BL/6J-YA/HeJ) were generated through successive

80 backcrosses, enabling the investigation of the contribution (if any) solely of the YA/HeJ.

C57BL/6J-YA/HeJ males were mated to C57BL/6J females and the resulting fetuses were

examined via PCR for the sex chromosomes and visual analysis of the gonads. Of the 23

Y-positive fetuses, 5 (22%) had at least one ovotestis, indicating that hermaphroditism is

a trait associated with the YA/HeJ chromosome.

Comparison of A/HeJ and BALB/cWt

Sequence

Hermaphroditism is also a trait of the BALB/cWt (Wt) inbred strain of mouse.

Originally characterized in 1975 (Whitten), subsequent analysis showed that it was nondisjunction of the Wt Y chromosome (YWt) primarily during the early embryonic

divisions that caused the disturbance in sexual differentiation (Beamer, Whitten et al.

1978; Eicher, Beamer et al. 1980; Bean, Hunt et al. 2001). The BALB/c and A strains of

mice are descendents of a common ancestor, Bagg Albino (Potter 1985). To investigate

if the YWt and YA/HeJ descended from the same Y chromosome and share a common

defect, we analyzed repeat sequences located on the short arm of the Y chromosome.

The GATA and GACA repeats, known as Bkm repeats, are subject to rapid evolution due

to a lack of selective pressure (Epplen, McCarrey et al. 1982). Comparison of Y-specific

banding patterns demonstrates dissimilarity between YWt and YA/HeJ, thus providing

evidence that these two Y chromosomes are not identical.

81 Segregation Behavior

Hermaphroditism on the A/HeJ strain could be caused either by a mutation on the

Y chromosome, missegregation of the Y, or a combination of both. To investigate

whether the YA/HeJ is nondisjunction-prone, the sex chromosome composition of mid- gestation C57BL/6J-YA/HeJ fetuses was analyzed in liver and fibroblast tissue. Similar

analysis in BALB/cWt found that Y chromosome hyperploidy was nearly identical in

individual fetuses, indicating segregation errors occurred primarily before the two tissue

types differentiated from one another (Bean, Hunt et al. 2001). Compared to C57BL/6J-

YA/HeJ males, there was not a significant increase in Y chromosome hyperploidy in liver

or fibroblasts from C57BL/6J-YA/HeJ hermaphrodites. There was a significant increase in

Y chromosome hyperploidy in C57BL/6J-YA/HeJ hermaphrodites compared to C57BL/6 controls, but only in liver. These results indicate two things: 1) Even though YA/HeJ is associated with hermaphroditism, missegregation of the YA/HeJ chromosome, while

increased over controls, is not responsible for the observed hermaphroditism and 2)

unlike the YWt, errors do not appear to be limited to the early cleavage divisions.

It is possible for a chromosome to be error-prone during meiosis, but not .

The meiotic behavior of the YA/HeJ was investigated during three time points: pachytene,

diakinesis/metaphase I and metaphase II. There was no significant increase in errors at

any of these time points in C57BL/6J-YA/HeJ animals compared to C57BL/6 controls.

Thus, YA/HeJ errors appear to be limited to mitosis.

82 Structural Analysis of the YA/HeJ Chromosome

The structural integrity of the YA/HeJ chromosome was investigated using a panel

of Y chromosome-specific FISH probes. Additionally, a probe that hybridizes to the

satellite repeats located at the centromeres of all mouse chromosomes—except the Y

chromosome—was utilized. The YA/HeJ chromosome, the Y chromosome from

BALB/cWt and two control Y chromosomes, BALB/cBy and C57BL/6J, were compared.

No obvious structural abnormality was detected in any of the four Y chromosomes

analyzed.

Morphological Aberrations of the A/HeJ Y Centromere

During the mitotic segregation analysis, it was noted that frequently there was a

“gap” between YA/HeJ chromatids (i.e., a space between sister centromeres). This phenotype was significantly increased in C57BL/6J-YA/HeJ hermaphrodites and males

compared to C57BL/6J controls in both liver and fibroblasts. Additionally, in individual

animals, the frequency of YA/HeJ chromatid gaps was not correlated with the level of Y

chromosome hyperploidy or gonad development (male vs. hermaphrodite). Therefore, it does not seem as if this gap phenotype causes either the increase in YA/HeJ missegregation or the increase in hermaphroditism. Evidence of a similar YA/HeJ centromere phenotype in meiotic cells was not seen.

Based on the gap phenotype and increased occurrence of hermaphrodites, we hypothesize that a mutation at or near the centromere affects both the sex determining properties and the segregation of the A/HeJ Y chromosome. Thus, this Y chromosome

83 serves as a good tool to investigate both centromere structure/function and sex

determining genes.

Disinfectants and Dysfunction: Solving the Mystery Breeding Problems in a New Colony

Our laboratory relocated from Case to Washington State University, moving four

inbred and thirteen mutant strains of mice. All stocks were breeding within a few

months; however, we began to notice a significant increase in reproductive maladies.

One set of studies required a large number of C57BL/6J timed pregnancies. According to

our previous experience and data from The Jackson Laboratory, approximately 70% of

C57BL/6J matings should result in a pregnancy. Within our first four months at WSU, the pregnancy rate was only 10.9%. Of the pregnancies that were maintained and used to obtain late gestation fetuses, a variety of defects were observed, including late fetal death, developmental acceleration/delay, and a variety of unusual developmental defects.

Additionally, we also noticed that in our C57BL/6J breeder cages, survival to weaning was significantly decreased compared to the breeding history at Case, and we were losing a number of pregnant breeder females to dystocia.

Changing the Environment

To improve reproductive performance, we systematically modified environmental conditions in the vivarium. We changed to the same diet used at Case, changed the light cycle and temperature and ruled out the presence of disrupting ultrasonic noises. All of these had a collective, positive impact on C57BL/6J pregnancy rates. The most notable

84 increase, however, came after limiting access to our colony to a single, dedicated member

of the vivarium staff. We therefore thought frequent disturbances, coupled with other

minor environmental factors, were the source of the poor reproductive performance.

Reproductive Problems Continue

Concurrently, another set of laboratory studies depended on superovulation of

young (4-6 week old) C57BL/6J females. By treating animals with exogenous hormones,

the number of eggs ovulated by a female can be increased. The technique was routinely

used by our laboratory at Case, with an average collection of 20-30 oocytes/female. At

WSU, however, animals showed little to no response to injections, despite switching

vendors and lots of hormones. Further, to collect embryos, we began mating

superovulated females. In addition to the decreased number of ovulated oocytes, we also

observed reduced egg quality (indicated by a high proportion of oocytes remaining

unfertilized) and more surprising, accelerated transport through the reproductive tract

(embryos prematurely entering the uterus).

Identifying the Source of the Reproductive Disturbances

A literature search was done to identify potential causes of accelerated transport through the reproductive tract. Interestingly, a citation was found that indicated the

pesticide methoxychlor could cause such a phenotype (Cummings and Perreault 1990).

Of the five investigators that house animals in the vivarium, three do toxicology research,

one specifically with methoxchlor. To investigate if it was a perpetual exposure to the

combination of chemicals present in the facility that was causing our breeding problems,

85 we analyzed cages for the presence of chemical contamination. Myriad compounds were present on the inside surfaces of “clean” cages, however none of them were those being used in toxicology research. After several rounds of analysis, it was noted that one compound, the disinfectant used throughout the mouse facility, was present in all samples.

The vivarium at WSU houses both specific pathogen-free (SPF) animals, such as ours, and conventionally caged animals; disinfectant was used liberally throughout the facility in an attempt to prevent cross-contamination. The disinfectant being used contained two quaternary ammonium compounds, n-alkyl dimethyl benzyl ammonium chlorides and didecyl dimethyl ammonium chloride. Times of known increased use

(specifically, two outbreaks of pinworm in non-SPF rooms) and decreased use (the dedicated staff member assigned to our colony was wary and thus sparingly used disinfectant directly around the animals) correlated with decreases and increases, respectively, in pregnancy rates.

Eliminating the Quaternary Ammonium Disinfectant

After the identification of the quaternary ammonium disinfectant as the probable cause of the observed breeding problems, two procedural changes were made in the vivarium. First, the cage wash cycle was made more stringent (no longer recycling water between loads, an additional wash and extended rinse times). This greatly reduced the amount of compounds present on cages, including the quaternary ammonium disinfectant; it did not, however, entirely eliminate the contamination. Thus, the quaternary ammonium disinfectant was removed from the facility and replaced with one

86 containing sodium chlorite. We continued to monitor cages and after approximately six

months, the level of detectable quaternary ammonium residue was consistently low.

Accidental Recontamination

A study was begun to directly test the effect of quaternary ammonium compounds

on mouse reproduction. A number of breeder cages were exposed to aerosolized disinfectant several times a week. These animals were housed in a dedicated room and the cages were washed separately from all other cages. Also, a purge cycle was run in the cage washer after every load of contaminated cages. Within months, however, breeding problems again became apparent in our colony. We analyzed cages washed both before and after a load of contaminated cages and found that despite all the changes in the wash protocol, the washer was still harboring and spreading quaternary ammonium residue. We immediately ended our exposure study.

87 Future Directions

Studies of the A/HeJ Y Chromosome

Our studies of the A/HeJ Y chromosome produced two seemingly separate observations. First, the centromere appears to be aberrant, as evidenced by the “gap” frequently noted between sister chromatids at metaphase. Second, contrary to expectation, the hermaphroditism associated with the YA/HeJ chromosome does not appear to result from loss of the Y chromosome. Experiments that address each of these findings are outlined below and, because I believe that the two findings stem from a common etiology, a set of experiments that considers these phenotypes in tandem is outlined.

While specifically investigating the YA/HeJ chromosome, the overall goal of the proposed studies is to use the YA/HeJ chromosome as a tool to address more global questions about centromere structure and function and the genetic control of sex determination.

Centromere

The Y chromosome centromere differs from all other human and mouse centromeres, as elements that characterize all other centromeres in the genome are greatly reduced or entirely missing (e.g., satellite repeats, CENP-B and CENP-A). As discussed in Chapter 2, the Y centromere has the characteristics of a centromere “on the edge;” it is easily mutated and inactivated. The sequence of this minimalist centromere has yet to be determined, but may provide insight to the essential elements of mammalian centromeres.

Segregation impaired chromosomes, like the A/HeJ Y chromosome, presumably have alterations in an essential component(s) and would further aid in the study of centromeres..

88 The YA/HeJ chromosome frequently displays a pronounced gap between sister

chromatids at metaphase. This phenotype suggests that cohesion between sister

centromeres is either not appropriately established or is prematurely lost. Obviously,

centromere cohesion is established, otherwise, the frequency of YA/HeJ missegregation

would be much higher. Thus, the most logical explanation for the observed phenotypic

abnormality is a reduction in the amount of bound at this centromere, e.g.

perhaps due to the loss of cohesin binding sites. This raises the possibility that, in

addition to needing the proper elements, a minimum number of copies of each element

must be present to create a properly functioning centromere. Chromatin

Immunoprecipitation (CHIP) analysis using antibodies to centromere-associated proteins such as cohesin and CENP-A, followed by Southern blot analysis, would allow for the identification of large deletions at the YA/HeJ centromere. Although not expanding the list of “known” centromeric elements, these studies might provide insight to the question of

“how much is enough” to create a functional centromere.

Sexual Differentiation

Over 2300 genes are expressed in a sex-specific manner in the developing mouse

fetus (Nef, Schaad et al. 2005). Sry (sex-determining region, Y chromosome) is thought

to be the “master switch”, that commits the gonad to develop as a testis (Gubbay,

Collignon et al. 1990; Koopman, Gubbay et al. 1991). Despite extensive research,

however, the complete genetic pathway of sex determination remains to be elucidated.

This may, in part, be a reflection of the fact that Sry is a rapidly evolving gene (Graves

2002).

89 The hermaphroditism caused by the A/HeJ Y chromosome could be due to

impaired interaction between Sry and downstream testis-determining autosomal (tda)

genes, or to insufficient or delayed Sry expression. Aberrations of both types have been reported previously. For example, C57BL/6J XY mice that carry the Sry gene from M. d. poschiavinus develop either ovaries or ovotestes but not normal testes (Eicher,

Washburn et al. 1982). This sex reversal is caused by both insufficient Sry expression and impaired interaction of the M. d. poschiavinus-derived SRY protein isoform with

C57BL/6J tda genes (Albrecht, Young et al. 2003). To determine if the aberrations in sexual differentiation observed in A/HeJ mice have a similar etiology, the timing and expression of Sry could be determined using a combination of RT-PCR and whole-mount in situ hybridization (WISH).

Concurrent investigations of downstream genes could also prove informative. Sry may be the initiator, but the expression of downstream genes is necessary for correct male sexual differentiation. Certainly genes known to be involved in sex determination

(e.g., Sox9, Fgf9 or Mis) should be studied. However, the use of expression arrays to identify significant changes in expression in C57BL/6-YA/HeJ animals by comparison with

C57BL/6J controls could lead to the discovery of genes not yet unknown to be involved

in this process. These studies would prove especially interesting if the expression levels

and timing of the A/HeJ Sry were comparable to controls, as this finding would

suggest aberrations in downstream signaling. Together, these analyses would help

elucidate not only the cause of hermaphrodites on the A/HeJ strain, but, more

importantly, provide new insight to the genetic control of male sexual differentiation.

90 Epigenetic Analysis

The studies outlined above consider the Y chromosome centromere defect and the

aberrations in sex determination that characterize the YA/HeJ chromosome as separate and

unrelated defects. I believe, however, that these phenotypes are related and share a common origin. The Sry gene is located on the very small p arm of the Y chromosome adjacent to the centromere. Thus, a centromere mutation (e.g., a deletion) that affects Sry

expression via a position effect provides a simple, logical explanation for both

phenotypic abnormalities.

The two tenets of this hypothesis (a centromeric deletion and epigenetic changes

in Sry) could be tested separately. Analysis to investigate a large deletion of centromeric

material could be done using CHIP for centromere-associated proteins and Southern blot

analysis. Testing the hypothesis that the A/HeJ Sry gene has been epigenetically altered

could be done by at least two methods, both focusing on the methylation status of CpG

islands. CpG islands are commonly found in promoter regions of genes, and changes in

methylation levels in these regions directly impact gene expression. Methylation of Sry

could be investigated using either methylation sensitive restriction enzymes (such as

HpaII and MspI) or the more recently developed technique of “bisulfate sequencing”

(Rakyan 2004 ref).

As stated above, the overall goal of the proposed studies is to shed light on two

important areas of genetic research—the mammalian centromere and sex determination.

Whether the A/HeJ Y centromere and sex differentiation phenotypes are viewed as

separate defects or as defects with a common etiology, future studies of the A/HeJ Y

chromosome have the potential to provide useful information for both fields of study.

91 Analysis of the Effects of Quaternary Ammonium Compounds on Reproduction

A growing body of data from experimental studies suggests links between environmental toxins and infertility. However, the applicability of these experimental studies to humans remains controversial. Human studies are confounded by the inherent difficulty of conducting exposure studies and the fact that daily exposures are not limited to a single toxin, as we are exposed to many compounds in our everyday lives.

Additionally, conventional methods of measuring the risk a chemical poses to humans are proving inadequate. Traditionally, toxicology studies have assumed that the relationship between the dose of a chemical and an adverse response is linear. However, recent studies of endocrine disrupting chemicals, chemicals that mimic or interfere with the actions of endogenous hormones, have revealed a “U” shaped dose-response curve

(reviewed in Welshons, Thayer et al. 2003). This suggests that — at least for some chemicals — small doses can have more severe outcomes than larger doses. In addition, studies of these chemicals have raised the concern that adverse effects that cause epigenetic changes can be transmitted to subsequent generations (i.e., transgenerational effects). Lastly, it is becoming increasingly evident that the fetus may be exquisitely sensitive to some chemical exposures. During development repair and detoxifying mechanisms are not yet fully in place and children and fetuses are sensitive to doses of chemicals that have no effect on a fully developed adult (Bern, 1992).

The question of how to properly investigate human exposures can start to be answered using animal models. Exposures can be carefully controlled and monitored and the resulting phenotypes measured for multiple generations. Chapter 3 of this dissertation describes adverse effects on the reproductive fitness of our mouse colony as a

92 result of prolonged exposure to a quaternary ammonium disinfect. The future studies

suggested below go beyond simply trying to explain this specific event, but attempt to aid

in unraveling the link between environmental toxins and infertility.

The disinfectant currently being investigated has two quaternary ammonium

compounds. Each compound could be separately used to treat animals, thus investigating

the effect of the components of the disinfectant. Using pure forms of the compounds a

wide range of doses can be delivered and a dose-response curve established. Finally,

dosing studies could be done at several developmental time points (e.g., in utero,

perinatally, after the onset of puberty, etc.) to determine the critical window(s) of

exposure.

The reproductive disturbances observed in our mouse colony appear to involve many reproductive organs; thus, it seems likely that quaternary ammonium compounds exert their effects by interfering with the endocrine system. Concentrations of endogenous hormones such as estradiol, , luetenizing hormone (LH), follicle stimulating hormone (FSH), and gonadotropin releasing hormone (GnRH) should be measured in blood samples from exposed and unexposed animals. Similarly, gene chips could be used to compare expression profiles in the pituitary and ovary of exposed and unexposed animals. As with dosing experiments, multiple time points should be investigated.

While understanding the impact of quaternary ammonium compounds on mouse fertility is interesting, a far more important question is the potential impact of these chemicals on humans. However, understanding the changes in hormone levels or organ function induced by these chemicals in an experimental model will enable researchers to

93 narrow the scope of their studies in humans and ask focused questions. Investigating a

full range of doses and establishing the true shape of a dose-response relationship at all

stages of development is key to establishing the safety of a chemical.

It has been proposed that human fertility is declining (e.g., Chandra 2005; Luoma

2005) and, despite the inherent difficulties of studies in humans, an increasing number of reports in the literature provide evidence of a link between environmental toxins and infertility in animals. Indeed, the weight of evidence is now too strong to ignore. We must move past the established methods of toxicology research and begin to see the whole picture of exposure, not only for ourselves, but for the generations to come.

94 Bibliography

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