Role of Pp1γ2 Binding Partners in Spermatogenesis and Sperm Function

ROLE OF PP1γ2 BINDING PARTNERS IN SPERMATOGENESIS AND SPERM FUNCTION.

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Shandilya Ramdas

December 2012

Dissertation written by Shandilya Ramdas B.S., Osmania University, 2002 M.S., Christian Medical College, 2005 Ph.D., Kent State University, 2012

Approved by

______, Chair, Doctoral Dissertation Committee Srinivasan Vijayaraghavan

______, Members, Doctoral Dissertation Committee Douglas W. Kline

______, Yijing Chen

______, Wen-Hai Chou

______. Soumitra Basu

Accepted by

______, Chair, Department of Biological Sciences James L. Blank

______, Associate Dean, College of Arts and Sciences Raymond Craig

ii TABLE OF CONTENTS

LIST OF FIGURES …………………………………………………………...iv

LIST OF TABLES …………………………………………………………...viii

ACKNOWLEDGEMENTS ……………………………………………………ix

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

MATERIALS AND METHODS.……………………………………………...29

RESULTS.………………………………………...... 38

DISCUSSION …………………………………...... 104

BIBLIOGRAPHY ………………………………………………...... 124

APPENDIX I ……………………………………………………………...….142

APPENDIX II ……………………………………………………………….....149

iii LIST OF FIGURES

Figure 1. Schematic representation of testis and various stages of

spermatogenesis occurring in the seminiferous tubule ……...... 2

Figure 2. Schematic representation of mechanisms that give rise to alternate

transcript variants …………………………………………………….. 8

Figure 3. A summary of spermatogenesis highlighting important events ……....10

Figure 4. Diagram depicting the structure of mouse and human sperm and

the ultrastructure of various regions of the sperm flagellum ………… 12

Figure 5. Diagrammatic representation of “9+2” microtubule arrangement

of the axoneme ………………………………………………………. 13

Figure 6. Differences in progressive and hyperactivated motility ……………... 15

Figure 7. Schematic depicting coupling of sulfolink column with peptide

against which the antibody was raised ……………………………….. 29

Figure 8. Map of pGEM – T Easy vector used for cloning of testicular

isoform messages …………………………………………………….. 33

Figure 9. Strategy to determine the role of P30 in epididymal

sperm maturation …………………………………………………….39-40

Figure 10. RSPH1 amino acid sequence depicting epitopes against which

antibodies were raised ……………………………………………… 40

Figure 11. Presence of RSPH1 isoforms in testis and sperm………..…………..41

Figure 12. Region-specific expression of P30 in the epididymis .…………...… 43

iv Figure 13. Third epitope against which a new RSPH1 antibody

was raised …………………………………………………….……..44

Figure 14. Validation of new RSPH1 antibody.………………………………...45

Figure 15. Presence of P30 in Rsph1 -/- mice ………………….…………….. 46

Figures 16-20. Purification and identification of P30 ……………………… 47-50

Figure 21. Co-elution of Rsph1 with PP1γ2 and its binding partners – I3,

sds22 and 14-3-3 ..………………………………………………… 51

Figure 22. Comparison of expression levels of PP1γ2 and its binding

partners – I3, sds22 and 14-3-3 between wildtype and

Rsph1 -/- mice ……………………………………………………... 52

Figure 23. Evidence that Rsph1 interacts with PP1γ2 and its binding

partners PPP1R11 (I3) and PPP1R7 (sds22)…………………..…... 53

Figure 24. Immunoprecipitation indicating I3 interacts with RSPH1………….54

Figure 25. Mouse CMUB116(IQUB) depicting the three epitopes

against which the anti-CMUB116 antibody was raised…………….56

Figure 26. Validation of anti-CMUB116 antibody.…………………………... 56

Figure 27. EST database demonstrating maximum expression

of CMUB116 in testis …………………………………………...... 57

Figure 28. Co-immunoprecipitation demonstrating the interaction between

RSPH1 and CMUB116 in mouse testis protein extracts…………....58

Figure 29. Validation of the antibodies for I2, I3 and sds22………………….61-62

v Figure 30. Amino acid sequence of PPP1R2 (I2) depicting the

epitopes against which antibodies were raised. ……...... ……..... 62

Figure 31. Validation of new C-terminus I2 antibody………...……………… 63

Figure 32. Presence of I2 in mouse testis (T) and sperm (S).……………….... 63

Figure 33. Schematic representation of alternate splicing of I2……….……... .65

Figure 34. Comparison of amino acid sequences of I2 isoforms……………... 65

Figure 35. Presence of transcript of alternate isoform of I2 in testis…..……... 66

Figure 36. Tissue Northern blot showing testis-specific expression

of unique isoform of I2……………………....…………………..... 67

Figures 37-38. Confirmation of presence of unique testicular isoform

of I2 by RT-PCR ……………….…………………………...... 68

Figure 39. Purification of I2 from mouse testis and sperm ……………...... 70

Figure 40. Identification and relative abundance of I2 by LC-MS ……………71

Figure 41. Schematic depicting alternate transcription start sites for I3…...... 72

Figure 42. Schematic representation of alternate isoforms of I3…..…………...73

Figure 43. Comparison of amino acid sequences of I3 isoforms ……………....73

Figure 44. Tissue Northern blot showing predominant expression

of alternate isoform of I3…………………………………………….74

Figures 45-46. Confirmation of presence of unique testicular isoform

of I2 by RT-PCR ………………………………………...……..75

Figures 47. Epitopes of an antibodies used in the identification

of alternate isoform of I3 ………………………………………...76-77

vi Figures 48-49. Validation of two new I3 antibodies……….……………....77-78

Figures 50-51. Purification and enrichment of testis isoform of I3….….… 79-80

Figure 52. Schematic representation of alternate splicing of sds2s……...... 80

Figure 53. Comparison of amino acid sequences of sds22 isoforms…….....82-83

Figure 54. Tissue Northern blot showing testis-specific expression

of unique isoform of sds22………………………………………82-83

Figures 55-56. Confirmation of presence of unique testicular isoform

of sds22 by RT-PCR………………………………………...83-84

Figure 57. Epitopes of an antibodies used in the identification

of alternate isoform of sds22…………………………………...... 84

Figures 58-59. Purification of testis isoform of sds22 from mouse

testis and sperm…………………………………………...... 86-87

Figures 60-65. Developmental expression of I2, I3 and sds22……………...88-91

Figures 66-67. Reduced expression of I2, I3 and sds22 in

Ppp1Cc -/- testis…………………………………………….92-93

Figure 68. DIC images of malformed sperm from Ppp1Cc2

rescue mice………………………………………………………..95

Figures 69-70. 2D-DIGE of A-line sperm ...... ………………………97-98

Figures 71-72. 2D-DIGE of E-line sperm …………………………………..98-99

Figure 73. Glycolytic enzymes identified from 2D-DIGE analysis………...... 101

Figure 74. Gluconeogenic enzymes identified from

2D-DIGE analysis…………………………………………………102

vii Figure 75. Schematic representing the localization and function of the

different kinds of CRISPs in male reproduction ………………….107

Figure 76. Schematic representing chemical genetics approach

to identify PKA substrates…………………………………….....120-21

viii LIST OF TABLES

Table 1. Genes essential for premeiotic development of germ cells ……………. 4

Table 2. Genes essential for meiosis during spermatogenesis …………………... 6

Table 3. Genes essential for spermiogenesis, epididymal maturation,

capacitation and fertilization ………………………………………….. 16

Table 4. Different subfamilies of PPPs ………………………………………… 18

Table 5. Primers used for genotyping Rsph1 knockout mice …………………....30

Table 6. 2D-DIGE spots with fold-differences and approx. molecular weights .. 85

DEDICATED TO MY PARENTS

x ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Vijayaraghavan, for his guidance and encouragement during my time in his lab. I am deeply indebted to his endeavors to provide a great environment to learn and challenge myself while trying to achieve my goal to complete my PhD. He was always there when I needed help in my work and at the same time allowed me to try and find answers in my own way. I would also like to thank him for his patience during my time in his lab and for also having the time to talk to about matters other than work. I would also like to thank Dr. Pilder who has had a very close association with our lab and in helping me with my work. I really appreciate the time and patience of the members on my dissertation committee – Dr. Kline, Dr. Chen,

Dr. Chou, Dr. Basu and Dr. Dutta. I would also like to thank Dr. Faber for his help with

DNA sequencing and Dr. Willard for her help with microsequencing. I would not have been able to complete this work without the support and encouragement of my colleagues, from whom I have learnt a lot. I was fortunate to have interacted with a lot of people, who have been part of this lab and members of this department. These people include Kim, Rumela, Lina, Ben, John, Pawan, David, Suraj, Vinay, Kurtis, Steve, Luis and the current members Nilam, Teja, Nida Sabyasachi, Dawn and Shuvalakshmi. I would like to thank my friends and roommates Siddharth, Parag, Manas, Sajid, Ashish,

Praveen and Amit for their support. Finally, I would like to thank my sister, brother-in- law and nieces for their constant support

Chapter 1

Introduction

1.1 Spermatogenesis

Mature spermatozoa capable of fertilizing an egg undergo various complex processes that begin in the male gonads and are completed in the female reproductive tract. Structurally competent haploid sperm are produced in the testis from diploid spermatogonial stem cells. This entire process comprises of tightly regulated steps including mitosis, meiosis and drastic morphogenesis known as spermiogenesis. This entire process takes place in the seminiferous tubule – the functional unit of testis. After spermiogenesis, the male gamete is transformed into a highly streamlined cell that has the primary purpose of delivering its “payload”, haploid nucleus, to the egg. After spermiogenesis, sperm are released into the lumen of the seminiferous tubule and transported to the epididymis via the rete testis by a process known as spermiation.

During epididymal transit, sperm undergo an additional process of maturation known as epididymal maturation during which they become motile and this form of motility is known as progressive motility. After ejaculation into the female reproductive tract, sperm undergo further maturation, which primes them for fertilization, known as capacitation.

Capacitation comprises of two unique phenomena -1) sperm undergo hyperactivation wherein, sperm exhibit a unique from of motility which is very different from progressive motility and 2) acrosome reaction.

1 2

Spermatogonial stem cell (SSC) (designated as Ao in rats and Ad in humans) present

adjacent to the germinal epithelium undergo mitosis to give rise to two daughter cells.

One enters spermatogenesis and the other remains as a SSC. The daughter cell that has

been “chosen” to undergo spermatogenesis undergoes further rounds of mitosis – six in

rats (1) and two or more in humans (2,3) that are tightly regulated. The final round of

mitosis, during which type B-spermatogonia gives rise to daughter cells, which are

Figure 1. Schematic representation of testis and various stages of spermatogenesis occurring in the seminiferous tubule (4).

DARSZON ET AL. Downloaded from

FIGURE 2. Diagram of the mammalian male reproductive organs and spermatogenesis. Left: spermatogenesis diagram. Spermatogenesis advances from the base to the lumen of the seminiferous tubule with

structurally and functionally essential nurse cells, called Sertoli cells. Spermatogonia (germinal stem cells) physrev.physiology.org reside in the basal compartment and proliferate. Primary spermatocytes (leptotene) differentiate from spermatogonia and transverse the blood-testis barrier formed by tight junction of adjacent Sertoli cells. Secondary spermatocytes (pachytene and diplotene) are formed by the first meiosis and converted into round spermatids by the second meiosis. Elongated spermatids are cells in the process of spermiation undergoing the remarkable morphological transformation of the nucleus and cell body. In the final process of spermiation, most of the cytoplasm will be removed (phagocytized by Sertoli cells) to form fully developed spermatozoa (the residual cytoplasm is known as cytoplasmic droplet). Middle: a seminiferous tubule and its cross-section. Newly formed spermatozoa are transported towards the epididymis through the lumen of the tubule (L). Right: testis and epididymis. The testis is filled with tightly packed seminiferous tubules; one of them is represented as a gray line on January 23, 2012 inside the testis. The epididymis consists of a single highly coiled tubules. This organ can be roughly divided into three portions: caput, corpus and cauda; sperm transit along these tubules is required for sperm maturation. not necessarily mean that the protein will be functionally II. MAMMALIAN SPERMATOGENESIS expressed in mature spermatozoa. The physiological rele- vance of the transcripts detected in mature spermatozoa is Spermatogenesis is the transformation of diploid spermato- still unclear and cannot be taken as proof that the proteins gonial stem cells into haploid spermatozoa that takes place they code for are present in spermatozoa. Therefore, dem- inside the seminiferous tubules (FIG. 2). It is a continuous, onstrating that an ion channel is functionally present in complex, and highly regulated process. Functional units mature spermatozoa requires proving either by controlled along the tubule’s epithelial compartments (basal and adlumi- immunological or proteomic strategies that the protein is nal) repeat a cycle that can be divided in four main phases: there, and showing that it is functional either electrophysi- mitosis, meiosis, spermiogenesis, and spermiation. In the ologically or using ion-sensitive dyes or proteins. Pharma- basal compartment, primary spermatogonia proliferate and cology and ultimately elimination of the specific ion channel after a species-specific fixed number of mitotic divisions from spermatozoa, in the species where it can be done (i.e., (253) differentiate into leptotene spermatocytes that are the null mice), will yield clear evidence of the role of the channel only cell type that transverses the blood-testis barrier into in sperm physiology. the adluminal compartment. Once in the adluminal compartment, cells differentiate to pachytene and then diplo- The authors are aware of their limitations and preferences, tene spermatocytes that undergo two rounds of meiotic di- which most likely have led to some biased proposals and vision to produce haploid spermatids. The process contin- regrettably to exclude some important contributions. Sev- ues with spermiogenesis in which spermatids experience a eral excellent reviews are provided, hoping they will com- strong morphological transformation including, among plement and balance the subject matter of this review (47, others, the formation of the acrosome, chromatin conden- 178, 418, 433, 548). sation, and the elimination of the excess cytoplasm (residual

Physiol Rev • VOL 91 • OCTOBER 2011 • www.prv.org 1307 3

committed to enter meiosis as preleptoene spermatocytes. Spermatogonial cells and meiotic and postmeiotic germ cells are located in different regions of the tubule.

Spermatogonial cells are located in the basal compartment of the tubule whereas meiotic and postmeiotic germ cells are located in the adluminal compartment. In the adluminal compartment, meiotic and postemeiotic germ cells are in close association with Sertoli cells (3,5). The germ cells in the adluminal compartment are separated from the basal compartment and do not receive any nutrition or do not come in contact with hormones from the blood stream, making them dependent on Sertoli cells for their survival and maturation (6). DNA synthesis and duplication occurs in preleptotene spermatocytes (7) and RNA synthesis during pachytene spermatocytes, which undergo meiosis to give rise to four haploid spermatids. A number of genes have been identified that play a vital role during spermatogenesis using knock out mouse models. These haploid spermatids undergo morphogenesis during a process called spermiogenesis.

Mouse models in male fertility research D Jamsai and MK O’Bryan 144

seminiferous tubules. In mice, spermatogonia begin to proliferate (as well as self-renew) and differentiate at ,4 days after birth and divide continuously through mitosis to give rise to spermatocytes. Premeiotic defects can lead to the complete disruption of spermatogenesis and result in a phenotype equivalent to that of the ‘Sertoli cell- only’ syndrome seen in ,15.7% of men with no sperm in their ejacu- lates.53 Several genes involving mouse spermatogonia self-renewal, apoptosis and cell cycle regulation have been shown to contribute to male infertility. Examples of such genes are listed in Table 1.

Meiotic defects Meiosis is a special cell division whereby diploid parental cells produce genetically diverse haploid sperm (or eggs). The reduction in chromosome number is achieved by one round of DNA replication followed by two successive rounds of chromosome segregation (meiosis I and II). Meiosis I involves the segregation/separation of homologous chromosomes from each other, whereas meiosis II involves the segregation of sister chromatids and therefore resembles mitosis.61 Male germ cells in the mouse testis enter meiosis in the second week of life. During meiosis, germ cells are termed spermatocytes. Spermatocytes pass through G1 and S phase and subsequently enter the meiotic prophase, during which time chromosome condensation and the formation of DNA double-strand breaks (DSBs) (leptotene spermatocytes), followed by the initiation of pairing (synapsis) between homologous chromosomes (zygotene spermatocytes) occur. The completion of synapsis of homologous chromosomes and the repair of DSBs, using homologous chromosomes as templates, occur from the midzygotene through the pachytene spermatocyte periods. The culmination of this process results in the reciprocal exchange of genetic information between homologues, and is completed through Figure 5 Example of a three-generation breeding scheme used to identify the midpachytene to diplotene periods.62 Genetic exchange takes place recessive mutations. To screen for recessive mutations, a three-generation through the formation of DSBs followed by a crossover (synapsis) of breeding scheme is required. Founder male mouse (referred to as generation genetic material between homologous chromosome pairs. This leads 0(G0)) of an inbred strain (for example, C57BL/6) is injected with ENU. The ENU- treated male is subsequently mated with wild-type females of a different inbred to the reassortment of maternal and paternal alleles and the produc-

strain (for example, CBA) to produce G1 offspring. G2 progeny can be produced tion of genetically diverged haploid sperm. The formation and repair from G1 littermate intercrosses or from G13wild-type CBA crosses (as shown of meiotic DSBs is a pivotal process that drives genetic diversity. here). Finally, G3 progeny are generated from G2 littermate intercrosses and/or G2 Due to the complexity of the meiotic process, a large number of females3G1 fathers. During each step of crossing, each progeny will have dif- genes are proposed to be involved in its regulation. Defects in this ferent combinations of chromosomes from the ENU-treated mouse strain and the process can lead to meiosis failure, the production of aneuploid strain used for subsequent outcrossing. These differences enable researchers to map the region containing the ENU-induced mutation causing phenotypic gametes and infertility. Furthermore, gamete aneuploidy can result 62 defects of interest (indicated by *). In this case, mutations are introduced into in embryonic death or developmental defects in the offspring. A list the C57BL/6 genome. CBA, cytometric bead array; ENU, N-ethyl-N-nitrosourea. of some crucial meiotic genes identified by the use of mouse4 models is shown in Table 2. Many of these genes are involved in the initiation of programmed DSB formation, meiotic recombination, DSB repair and

Table 1. Genes essential for premeiotic development of germ cells (adapted from Ref 8). Table 1 Examples of genes essential for premeiotic germ cell development implicated by mouse model studies Gene Proposed function Knockout phenotype Fertility status Reference

Etv5 (Erm) (Ets variant gene 5) Transcription factor Azoospermia; failed to maintain spermatogonia Male infertility 54 Bax (BCL2-associated X protein) Regulation of apoptosis Premeiotic germ cell arrest Male infertility 55 Pi3k (phosphoinositide-3-kinase) Phosphatidylinositol 39-kinase signaling Impaired spermatogonia proliferation and Male infertility 56 pathway increased apoptosis of spermatogonia Nanos2 (Nanos homolog 2 Germ cell differentiation Germ cell apoptosis and complete loss of Male infertility 57a,58 (Drosophila)) spermatogonia Ddx4 (Vasa)(DEAD(Asp–Glu–Ala– Germ cell proliferation and differentiation Impaired premeiotic germ cells differentiation and Male infertility 59 Asp) box polypeptide 4) increased apoptosis of spermatogonia Dazl (Deleted in azoospermia-like) Germ cell proliferation and differentiation Azoospermia; few spermatogonia enter meiosis, Male/female infertility 60 and those that do fail to proceed beyond pachytene

a Transgenic mouse model.

Asian Journal of Andrology 1.2 Spermiogenesis:

During this process haploid spermatids undergo differentiation to form elongated

spermatids and eventually structurally competent male germ cells known as spermatozoa.

The differentiation process involves formation of structures that are vital to sperm

function. These include the head, acrosome and the flagella. Apart from these visibly

unique structures sperm also start to express vital receptors and ion channels that

facilitates further maturation. During the formation of this highly streamlined cell, the

nucleus becomes elongated and then highly condensed, mitochondria are tightly coiled

around the midpiece of the flagellum and excess cytoplasm is shed, which remains

attached to sperm as a cytoplasmic droplet (9). In spermatids containing highly compact

nucleus, histones are replaced by transition proteins and then finally by protamine.

Spermiation follows spermiogenesis transport sperm from the lumen of the seminiferous tubule to the epididymis for further maturation. During spermiation, the cytoplasmic droplet is shed and secretions from the Sertoli cell transport sperm from the testis to the epididymis (9).

A precise and well-coordinated process of gene and protein expression is required for spermatogenesis to occur successfully. This is achieved by: 1) synthesis of male germ cell-specific proteins from alternate spliced transcripts, 2) storage of transcripts and minimal transcription during postmeiotic stages, 3) stage-specific translation of stored transcripts and 4) transcription and translation occurring independently of each other

(10).

Table 2. Genes essential for meiosis during spermatogenesis (adapted from Ref 8).

Table 2 Examples of genes essential for meiosis implicated by mouse model studies

Gene Proposed function Knockout phenotype Fertility status Reference

Spo11(Sporulation protein, meiosis-specific, SPO11Programmed homolog DSB formation Impaired DSB formation and recombination initiationMale/female infertility 63

(S. cerevisiae)) a Mei1(Meiosis defective 1) Programmed DSB formation Impaired DSB formation and recombination initiationMale/female infertility 64

Atm(Ataxia telangiectasia mutated homolog (human)) DSB sensing and repairing Impaired chromosomal synapsis and chromosome65 fragmentation Male/female infertility

Dmc1(Dosage suppressor of mck1 homolog, meiosis-specificMeiotic recombination Impaired chromosomal synapsis Male/female infertility 66

homologous recombination (yeast))

H2afx(H2A histone family, member X) Assembly of specific DSB repairMeiosis arrested at pachytene stage Male infertility/female subfertility 67

complexes

Trip13(Thyroid hormone receptor interactor 13)Meiotic recombination and DSB repair Impaired DSB repair Male/female infertility 68

Mlh1(MutL homologE. coli1)) ( Mismatch repair Impaired meiotic recombination Male/female infertility 69

Mlh3(MutL homologE. coli3)) ( Mismatch repair Impaired chromosomal synapsis Male/female infertility 70

Pms2(Postmeiotic segregation increasedS. cerevisiae 2)) ( Mismatch repair Impaired chromosomal synapsis Male infertility 71

Msh4(MutS homologE. coli4)) ( Mismatch repair Impaired chromosomal synapsis Male/female infertility 72

Msh5(MutS homologE. coli5)) ( Mismatch repair Impaired chromosomal synapsis and synaptonemalMale/female complexes infertility 73

formation

Exo1(Exonuclease 1) Mismatch repair Dynamic loss of chiasmata and apoptosis Male/female infertility 74

Cdk2(Cyclin-dependent kinase 2) Cell cycle regulation Meiosis arrest at prophase I and atrophy of the testes and ovaries Male/female75 infertility

Ccna1(Cyclin A1) Cell cycle regulation Meiosis prophase I arrest and increased germ cell apoptosis Male infertility76

Fkbp6(FK506-binding protein 6) Homologous chromosomes synapsis Meiosis arrest at pachytene stage Male infertility 77

Psmc3ip(Hop2, Tbpip) (Proteasome (prosome, macropain)Homologous chromosomes synapsis Meiosis arrest Male/female infertility 78

26S subunit, ATPase 3, interacting protein)

Dnmt3l(DNA (cytosine-5-)-methyltransferase 3-like) DNA methylationMeiosis arrest and epigenetic defects Male infertility; heterozygous79,80 progeny asiadM O’Bryan MK and research Jamsai fertility D male in models Mouse of homozygous females die by

midgestation

Siah1(Seven in absentia 1A) Protein ubiquitination and degradation Meiosis prophase I arrest Male infertility/female subfertility 81

Prdm9(Meisetz) (PR domain containing 9) Histone H3 methyltransferase thatImpaired chromosomal synapsis and sex body formation Male/female82 infertility

controls epigenetic events required for

meiotic prophase

Rec8(Rec8 homolog (yeast)) Synaptonemal complex formation Abnormal synaptonemal complexesMale/female infertility 83

Sycp1(Synaptonemal complex protein 1) Synaptonemal complex assembly,Impaired meiotic chromosomal synapsis Male/female infertility 84

recombination, and XY body formation

Sycp2(Synaptonemal complex protein 2) Synaptonemal complex assemblyImpaired and axial element formation Male infertility/female subfertility 85

chromosomal synapsis

Sycp3(Synaptonemal complex protein 3) Axial/lateral elements and synaptonemalImpaired axial/lateral elements and synaptonemalMale complexes infertility/female subfertility 86

complex formation and chromosomalformation

synapsis

Syce1(Synaptonemal complex central element protein 1) DSB repair Impaired DSB repair Male/female infertility 87

Syce2(Synaptonemal complex central element protein 2) SynaptonemalImpaired complex chromosomal formation, synapsis and sex body formation Male/female88 infertility

homologous recombination and DSB

repair

Smc1b(Structural maintenance of chromosomes 1B) Segregation ofImpaired chromosomes sister chromatid cohesion formation andMale/female chromosomal infertility 89 sa ora fAndrology of Journal Asian synapsis a Eif4g3(Eukaryotic translation initiationc, 3) factor 4 Translational regulation Meiotic arrest; spermatocytes failed tovia exitG2/MI prophaseMale infertility 49

transition

a Chemical-mutagenized point mutant mouse models.

Abbreviations: DSB, double-strandE. coli,Escherichia break; ;S. coli cerevisiae,Saccharomyces cerevisiae.

145

1.3 Regulation of spermatogenesis:

Spermatogenesis is an extremely well coordinated and tightly regulated process.

This level of regulation is required to accommodate the changing patterns and precise

initiation of gene expression that is required for the formation of mature sperm. This is achieved at three different levels: intrinsic, interactive and extrinsic (11).

Germ cell gene expression at the intrinsic level begins at transcription, then at translation and finally at the post-translational level. These three levels of regulation are highlighted by the fact that many unique genes and variants are expressed during spermatogenesis and their expression is stage specific and developmentally regulated.

Germ cell specific transcripts and proteins are produced in three major ways: 1) expression of germ-cell specific somatic homologs that are expressed either in somatic cells or in somatic and germ cells, 2) expression of unique genes that have no other homologs and 3) expression of alternate transcripts (10). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) is a glycolytic enzyme and is considered to be a housekeeping gene. Gapdh is inactivated during the meiotic phase of spermatogenesis and a germ cell- specific Gapdh-s isoform is activated during the postmeiotic phase (12,13). GAPDH-s varies from the somatic homolog only in the N-terminal region of the protein (11).

Another somatic isoform of a glycolytic enzyme, phosphoglycerate kinase-1 (Pgk1) is located on the X chromosome and is inactivated during the meiotic phase (14). A germ cell specific isoform Pgk2 is activated to compensate for this during the meiotic phase

(11). Apart from substituting for the somatic homolog during spermatogenesis and in sperm function, these homologs could have other structural and functional properties.

The presence of additional amino acid residues could play a role in their localization or their interaction with other molecules.

During spermiogenesis unique germ cell genes are expressed. One group of

proteins, comprising of transition nuclear proteins (Tnp1 and Tnp2) and protamine (Prm1

and Prm2) (11), are expressed at postmeiotic stages and play important roles during

remodeling of the sperm head and nuclear condensation. Germ cell specific proteins are

also synthesized from alternate transcripts. This results in proteins that are different from

Wang et al.their somatic isoforms in size and/or sequence. Page 11

Wang et al. Page 11 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 2. Pervasive tissue-specific regulation of alternative mRNA isoforms Rows represent the 8 different alternative transcript event types diagrammed. Mapped reads supporting expression of upper isoform, lower isoform or both isoforms are shown in blue, red, and gray, respectively. Columns 1-4 show numbers of events of each type: (1) supported by cDNA and/or ESTFigure data; 2. (2) Pervasive with 1tissue-specific isoform supported regulation by mRNA-SEQ of alternative reads; mRNA (3) isoforms with both isoforms supportedRows by reads; represent and (4) the events 8 different detected alternative as tissue-regulated transcript event (Fisher's types exact diagrammed. test) Mapped reads at an FDR of 5%. Columnssupporting 5-6 expression show: (5) theof upperobserved isoform, percentage lower ofisoform events orwith both both isoforms isoforms are shown in blue, detected that were red,observed and gray, to be respectively. tissue-regulated; Columns and (6)1-4 the show estimated numbers true of percentageevents of each of type: (1) supported tissue-regulated isoformsby cDNA after and/or correction EST data; for power (2) with to detect 1 isoform tissue supported bias and forby mRNA-SEQthe FDR (Fig. reads; (3) with both S3). For some eventisoforms types, ‘common supported reads’ by reads; (gray and bars) (4) wereevents used detected in lieu as of tissue-regulated (for tandem 3 (Fisher's exact test) UTR events) or in ataddition an FDR to of ‘exclusion’ 5%. Columns reads 5-6 for show: detection (5) the of observed changes percentagein isoform oflevels events with both isoforms between tissues. detected that were observed to be tissue-regulated; and (6) the estimated true percentage of tissue-regulated isoforms after correction for power to detect tissue bias and for the FDR (Fig. S3). For some event types, ‘common reads’ (gray bars) were used in lieu of (for tandem 3 UTR events) or in addition to ‘exclusion’ reads for detection of changes in isoform levels between tissues.

Nature. Author manuscript; available in PMC 2009 May 27.

Nature. Author manuscript; available in PMC 2009 May 27. 9

Figure 2. Schematic representation of mechanisms that give rise to alternate transcript variants ( adapted from Ref 15).

Apart from the mechanisms listed above, use of an alternative start site

(alternative first exon) and use of an alternative last exon are two more ways in which alternate transcripts are generated. The catalytic subunit (Cα) of PKA is present in two isoforms - Cα1 and Cα2. Both these isoforms are expressed in testis during spermatogenesis but Cα2 is expressed only from the pachytene spermatocyte stage of spermatogenesis. During this stage, an alternate promoter in the first intron of Cα gene is activated and an alternate variant is transcribed using an alternate start site. Cα2 has a distinct seven amino acid N-terminus compared to a fourteen amino acid N-terminus of

Cα1 (16). Germ cell specific isoforms of angiotensin-converting enzyme (ACE) (17) and

Ca2+/calmodulin-dependent kinase IV (CaMKIV) (18) are generated from transcripts that are synthesized from alternate start sites. Another mechanism utilized to generate alternate transcripts is by the utilization of alternate polyadenylation (poly (A)) site. This is an important mechanism used by germ cell specific transcripts that need to be stored and translated during a later stage in spermatogenesis (19). An example of this is seen in two germ cell specific isoforms of the cation-dependent mannose-6-phosphate receptor

(11).

The interactive and extrinsic modes of regulation help in relaying signals from hormones. Interactive mode involves the communication that occurs between Sertoli cells and the developing cells (11). Gene expression changes in Sertoli cells plays an important role in regulation of spermatogenesis. Testosterone produced by the Leydig cells is a

major component of extrinsic mode of regulation. Luetenizing hormone (LH) stimulates

testosterone production by Leydig cells and testosterone exerts its action by binding to

androgen receptor (AR) expressed on Sertoli cells and myoid cells and hence making it

important for spermatogenesis (11,20).

8 10 13 15

Meiotic divisions al et Grootegoed A. J. 336 I and II

B PL Early Mid Late Round/Elongating Condensing/Condensed Pachytene Steps 1–11 Steps 12–16

Mitotic cell cycle control Chromatin reorganization: Meiotic cell Chromatin reorganization: and entry into meiosis homologous chromosome pairing cycle control histone-to-protamine transition and meiotic recombination Removal of nuclear and cytoplasmic proteins

X and Y gene transcription Re-expression of X and Y genes

Protamine gene Protamine mRNA transcription translation

Switches to expression of spermatogenesis-specific genes Support by Sertoli cells: transmission of testosterone and FSH signalling, generation of local signals, maintenance of spermatogenic microenvironment, and direct structural interaction with the developing germ cells Figure 1. Schematic presentation of several events during spermatogenesis in the mouse. The developmental series of events that leads to the formation of spermatozoa requires pronounced support by Sertoli cells and a strict control of gene expression in the spermatogenic cells. This control of gene expression will evidently show dierent properties during the mitotic, meiotic and post-meiotic phases of spermatogenesis. These phases, however, form a continuum. This is most evident forthetransitionofmeiotic spermatocytes into post-meiotic spermatids, since spermatocytes already express a number of genes encoding proteins that are essential for sperm function rather than meiotic events. Spermatogonia may have similar foresight. The spermatogenic meiotic divisions result in the generation of haploid spermatids, which start out as round cells but then begin±with a long and pronounced cellular dierentiation process±to become spermatozoa. This process of spermiogenesis takes approximately 3 weeks. The dierentiation into the highly specialized sperm cells is one of the most remarkable cell developmental processes that can be observed in biological systems, involving phases of acrosome development, nuclear elongation and condensation, the formation of middle piece and tail, and the reduction of cytoplasmic volume. The cell types shown are spermatogonia B, pre-leptoteneFigure spermatocytes 3: A summary (PL), pachytene (early,of spermatogenesis mid and late) primary spermatocytes, highlighting and spermatids important during subsequent events steps of(adapted their develop ment.from The transient transcriptional inactivation of the X and Y chromosomes in spermatocytes, and the overall silencing of gene transcription upon histone-to-protamine transition in condensing spermatids,Ref is indicated21). by the dashed lines. FSH follicle-stimulating hormone.

1.4 Spermatozoa Structure

A mature spermatozoon is made up of two major components – the head,

containing the nucleus and the tail, the motility apparatus required to deliver the nucleus

to the egg in the female reproductive tract. The head and tail are held together by the connecting piece.

The head contains highly condensed chromatin, acrosome and extremely little amount of cytoplasm. The tail, beginning from the connecting piece, is divided into the midpiece, principal piece and the end piece regions successively. The main components of the tail are the axoneme, mitochondrial sheath (MS), outer dense fibers (ODF) and the fibrous sheath (FS). The axoneme contains a “9+2” complex of microtubules that extend the full length of the flagellum and forms the core. The ODF envelops the axoneme and is present from the connecting piece to the posterior portion of the principal piece. ODF is a characteristic feature of mammalian spermatozoa and its presence confers a “9+(9+2)” conformation in the mid-piece and most of the principal piece. The MS lies immediately adjacent to the ODF in the midpiece of the flagellum and contains tightly coiled mitochondria and the FS surrounds the ODF in the principal piece. The axoneme (axial filament complex) is made up of a central pair of microtubules (A and B microtubules) surrounded by nine outer microtubule doublets. This arrangement of microtubules gives it the “9+2” appearance, which can be observed in flagella and cilia of most plant and animal species (22,23). Sperm motility is the result of the active sliding of microtubules by axonemal dyenins. Axonemal dyenins are attached to the outer microtubule doublets

(A-microtubule) and based on the position of attachment to the microtubules they are classified as outer and inner dyenin arms (24). Another important structure that is attached to the outer microtubule doublet (A-microtubule) is the radial spoke (RS), which

is composed of a stalk and a head. RS is attached to the outer microtubule pair via the stalk and the head interacts with the central pair (CP) of microtubules.

Figure 4. Diagram depicting the structure of mouse and human sperm and the ultrastructure of various regions of the sperm flagellum (Ref 4).

526 Inaba

Figure 2 Internal structure of sperm flagella. (A) Comparison of human sperm flagella and primitive sperm flagella. In human sperm, the axoneme is surrounded by ODFs, mitochondria, and plasma membrane, whereas in the principal piece, it is surrounded by ODF, a FS and a plasma membrane. In sperm from sea urchins, tunicates and teleosts, the axonemes are simply surrounded by a plasma membrane. (B) Substructures comprising flagellar axonemes. Axonemal structures are well conserved among invertebrates, lower vertebrates and mammals. and betweenFigure the 5. protein Diagrammatic with an AKAP representation domain RSP3 and of central “9+2” microtubulecontribute to thearrangement elastic resistance of the involved in the conversion of apparatus AKAP-binding protein AAT-1 might mediate RS/CP appar- microtubule sliding into axonemal bending. Recent cryoelectron tom- atus interaction.axoneme (adapted from Ref 24). ography as well as previous thin section observation have demon- Dynein regulatory complex (DRC) is a structure observed at the strated the relationship between DRC and nexin links and structures junction between RSs and inner arm dyneins (Huang et al., 1982; inside the doublet microtubules (Tam and Lefebvre, 2002; Nicastro PipernoTheet alsliding., 1992; ofGardner axonemalet al., 1994dyenins). DRC and is the believed regulation to et brought al., 2005, 2006about; Sui by and the Downing, RS and 2006 CP; Heuser et al., 2009). both anchor inner arm dynein and functionally connect outer and inner armscomplex and RSs. brings From mutantabout pf2theand bendingpf3, seven of polypeptidesthe flagellum, which is translated into motility. In (29–192 kDa) have been identified as DRC components (Gardner Marine invertebrate sperm: a et al., 1994). Some of the components of DRC, such as a homolog model system for studying of growth-arrest-specificChlamydomonas gene, productRS is made PF2, have up been of at characterized least 23 polypeptides which play important roles in (Rupp and Porter, 2003). A protein from Trypanosoma brucei, flagellar proteins and 2+ CMF70,Ca which /calmodulin is a homolog and of a cAMP human spermsignaling flagellar (24) protein. Proteinsmechanisms containing the isoleucine of flagellar-glutamine motility NYD-SP28, has an ortholog in Chlamydomonas and may be a com- in metazoa ponent(IQ) of DRC motif (Zheng (25et), al .,A 2006-kinase; Kabututu anchoringet al., 2010 protein). Further- (AKAP) domain (26) and the regulatory more, interdoublet or nexin links are present as connective Sperm from marine invertebrates have long been used to study the structures between each doublet microtubule and are thought to molecular structure of flagellar axonemes and flagellar motility subunit (RII) alpha (27) have been identified.

1.5 Epididymal Maturation and Capacitation

Epididymal maturation is a process that occurs only in mammals. Sperm exiting

the testis and entering the epididymis are immotile. They attain progressive motility

during epididymal transit. The epididymis can be divided into three major segments –

caput (head), corpus (body) and cauda (tail). Sperm from testis are transported to the

caput region of the epididymis, then transported along the corpus and attain progressive motility in the caudal epididymis. Sperm during epididymal transit interact with proteins that are synthesized and secreted in a region-dependent manner from the epididymal epithelium. A series of biochemical, physiological and morphogenic changes occur that result in progressive motility. Significant changes occur in the sperm plasma membrane composition characterized by changes in phospholipds and amounts of cholesterol. These changes enable the formation of lipid rafts comprising of multimeric signaling complexes required for sperm function (28). Other important changes that occur which are essential for attaining motility is change in intracellular concentration of Ca2+, pH and cyclic adenosine mono-phosphate (cAMP) (29). During epididymal maturation, intracellular pH and cAMP levels increase and there is a decrease in intracellular Ca2+ levels. These changes are thought to bring about a change in protein phosphorylation that is essential for sperm motility (29).

Capacitation is the final process of sperm maturation that enables successful fertilization. During this process sperm attain the “capacity” to fertilize an egg and this occurs in the female reproductive tract. Sperm undergo biochemical and morphological changes, which include: 1) efflux of cholesterol, 2) increased levels of cAMP due to increased activity of adenylyl cyclase, 3) an increase in protein tyrosine phosphorylation levels, 4) increase in intracellular Ca2+ and pH and 5) changes in the biochemical properties of the sperm plasma membrane (30). These changes result in an increase in the fluidity of the sperm plasma membrane and alter the levels of intracellular messengers.

As a result of which, sperm undergo hyperactivated motility, achieve their ability to bind

DARSZON ET AL.

are subfertile as their spermatozoa show spontaneous AR the mechanism of how Ca2ϩ controls the form of flagellar alteration (334). bending is not fully understood. Pharmacological evidence indicates that CaMKII and/or CaMKIV is involved in this 2. Flagellar form modulation by Ca2ϩ process in mammalian sperm (269, 344). Recently, a novel Ca2ϩ-binding protein, named calaxin, was identified as an It is known that, in an appropriate medium supplied with axoneme component in sea squirt sperm (365). Bioinfor- ATP, the sperm flagellum can be maintained motile after matics revealed that calaxin belongs to the neuronal cal- removal of the plasma membrane by detergent treatment cium sensor protein family. Immunoelectron microscopy (213). With the use of demembranated sea urchin sperm, and biochemical analysis showed that it interacts with the the importance of Ca2ϩ for sperm flagellar form was first dynein heavy chain at the outer arm dynein in a Ca2ϩ- reported in 1974. The curvature of sperm swimming trajec- dependent manner. These results suggest that calaxin may tories became larger as the Ca2ϩ concentration was elevated be important for flagellar form regulation by Ca2ϩ (365). (73), due to the increase in flagellar asymmetry (71). These discoveries led to a central dogma that the degree of flagel- C. Hyperactivation 15 lar beat asymmetry is determined by the global Ca2ϩ con- 2ϩ centration. The first Ca imaging of the moving sperm In mammals, spermatozoa become motile when ejaculated flagellum was performed in 1993 using hamster spermato- into the female genital tract. The ejaculated spermatozoa 2ϩ zoa (493). This experiment revealed that the [Ca ]i of show symmetric flagellar beating with low curvature and hyperactivated spermatozoa was higher than that of acti- swim progressively with an almost straight path (FIG. 4). Downloaded from to the zona pellucidavated and spermatozoa are primed (see to undergo sect. IVC acrosome). Hyperactivated reaction sperm – the hallmarkThis swimming pattern is called “activated motility.” Fur- motility occurs during capacitation (see sect. IVF) and is thermore, a subpopulation of sperm recovered from the generally characterized by high amplitude and asymmetric features of capacitation (31). oviduct shows vigorous asymmetric flagellar beating with flagellar bends in nonviscous media (FIG. 4). However, we large amplitude and high curvature in a standard low-vis- 2ϩ now know that the relationship between [Ca ]i and flagel- cosity medium, which is called “hyperactivated motility.”

lar asymmetry is not always proportional, as described later Hyperactivation is an essential process for spermatozoa to physrev.physiology.org in sea urchin and sea squirt (ascidians) spermatozoa (465, successfully fertilize oocytes, and Ca2ϩ is a fundamental 562) (see sect. VI). Although several efforts have been made, regulatory factor; it is not known how it is triggered under physiological conditions (reviewed in Ref. 488). Possibly distinct factors such as bicarbonate (sect. IVD), progester- Progressive Hyperactivated Motility Motility one (sect. IVE), or a temperature decrease (sect. IVG) tran- siently induce hyperactivation at different sites and occa- sions while the spermatozoa search for the oocyte. on January 23, 2012

Ca2ϩ is a fundamental regulatory factor for sperm hyperactivation. With the use of demembraneted bull spermatozoa, it was demonstrated that Ca2ϩ but not cAMP can induce hyperactivation (259). For instance, 80% of spermatozoa show hyperactivation at 400 nM Ca2ϩ. As already 2ϩ mentioned, [Ca ]i is higher in hyperactivated hamster spermatozoa (100–300 nM) than in activated spermatozoa Figure 6. Differences in progressive and hyperactivated motility (Ref 4). (10–40 nM) (493). Ca2ϩ channels at the plasma membrane 2ϩ (CatSper) and from internal Ca stores (IP3R and RyR) seem involved in achieving hyperactivation (see below). In 2ϩ 2ϩ addition to Ca channels, sperm [Ca ]i homeostasis is 2ϩ A re-occurring theme during spermatogenesis and one that is vital to controlledsperm by several types of Ca transporters (552). A plasma membrane Ca2ϩ-ATPase 4 (PMCA4), localized in the principal piece of the flagellum, is crucial for sperm function is phosphorylation.FIGURE 4. TheProperties interplay and physiological between rolesprotein of hyperactivation. kinases (PKs) andfunction protein since its elimination affects sperm motility (459) A: activated (left) and hyperactivated (right) flagellar shape and motility direction (arrows) displayed by spermatozoa in nonviscous ex- and hyperactivation (395) resulting in male infertility. Mi- phosphatases (PPsperimental) determines media. the Activated phosphorylation spermatozoa advancestatus of showing cell’s a proteome. sym- tochondrial A protein abnormalities found in PMCA4-deficient sper- metric flagellar bend, while hyperactivated spermatozoa tumble in matozoa (395) suggest Ca2ϩ overload due to defective Ca2ϩ one place and do not advance efficiently due to an asymmetric extrusion. can be phosphorylatedflagellar at beatingmultiple pattern. residuesB: importance and this ofplays hyperactivated an important sperm role in modulating motility: i) To advance in highly viscoelastic fluids in the female genital 1. Physiological roles of hyperactivation its function (activatetract or more inactivate effectively an than enzyme) activated or sperm, determinesii) to detach its spermato- localization within a cell. zoa from the isthmus reservoir and advance towards the ampulla (site of fertilization), and iii) to facilitate sperm penetration through Hyperactivated spermatozoa are less progressive than acti- the cumulus matrix and the zona pellucida. vated spermatozoa in low viscous media. Some hyperacti-

Physiol Rev • VOL 91 • OCTOBER 2011 • www.prv.org 1319

Table 3 Examples of genes essential for spermiogenesis, epididymal maturation, capacitation and fertilization implicated by mouse model studies

Gene Proposed function Knockout phenotype Fertility status Reference

Spem1(Sperm maturation 1) Spermatid cytoplasmic removal Deformed sperm head and tail structure and impaired spermiogenesisMale infertility 144

Gopc(Golgi-associated PDZ and coiled-coilAcrosome biogenesis Fragmented acrosomes; globozoospermia (round-headed spermatozoa)Male infertility 145

motif containing)

Prm1andPrm2(Protamine 1 and 2) Sperm chromatin condensationHaploinsufficiency effect; sperm derived from spermatogenic cellsChimera with male/female one copy of90 the

mutated allele are immotile and unable to fertilize eggs infertility

Tnp1(Transition protein 1) Sperm chromatin condensation Subtle sperm morphology abnormalities and reduced motility Male subfertility 91

Tnp2(Transition protein 2) Sperm chromatin condensation Impaired chromatin condensation, sperm head abnormalities and reduced sperm motility92,93 Male subfertility

Tssk6(Sstk) (Testis-specific serine kinase 6) Chromatin remodelingAbnormal sperm head morphology, reduced DNA compactionMale in spermatozoa infertility and146

impaired sperm motility

Cadm1(Tslc1) (Cell adhesion molecule 1) Cell adhesion, differentiation and apoptosis Spermatid maturation arrest, degeneratedMale infertility and sloughed147,148 off into the lumen

Pvrl2(Nectin) (Poliovirus2 receptor-related 2) Cell adhesion Abnormal sperm head and sperm tail midpiece structure, impairedMale zona infertility binding, and149 lack

of oocyte penetration

Csnk2a2(Casein kinase 2, alpha primeSperm head morphogenesis Oligospermia (low sperm counts) and globozoospermia Male infertility 150

polypeptide)

Rara(Retinoic acid receptor, alpha)Ligand-induced transcription factorSloughing of immature germ cells in the lumen of seminiferousMale tubules infertility 151 a Pafah1b1(Lis1) (Platelet-activating factorAcrosome and tail biogenesis Impaired acrosome and tail formation; the basal tail cuff at theMale point infertilityof insertion but100 fail to

acetylhydrolase, isoform 1b) add axonemes to elongate the tail structure

Meig1(Meiosis-expressed gene 1) Sperm tail biogenesis,Absent assembly sperm of flagella, the abnormal sperm head, disrupted microtubularMale infertility organelle essential 101

axonemes/flagella for sperm head and flagella formation a Tekt2(Tektin-t) (Tektin 2) Sperm tail biogenesis, dynein armBending formation of thein sperm flagella and marked defects in motility and primary ciliary dyskinesia108 Male infertility

the axoneme

Adcy10(sAC) (Adenylate cyclase 10) Sperm motility Normal sperm counts and morphology with severely impaired motility Male infertility 152

Gapds(Glyceraldehyde-3-phosphateGeneration of energy supply for sperm motility Normal sperm counts and morphology with impairedMale infertility motility (loss of forward 117 progression

dehydrogenase, spermatogenic) motility) O’Bryan MK research and Jamsai fertility D male in models Mouse

Pgk2(Phosphoglycerate kinase 2) Generation of energy supply for sperm motility Impaired progressive motility Male subfertility 116

Agfg1(Hrb) (ArfGAP with FG repeats) Acrosome 1 biogenesis Globozoospermia Male infertility 153

Pick1(Protein interacting with C kinase 1) Vesicle trafficking fromFragmented the Golgi acrosomesapparatus to Globozoospermia Male infertility 154

the acrosome

Vdac3(Voltage-dependent anion channel 3) Ion transport for sperm tail formation Normal sperm counts, abnormal sperm tail structure and155 markedly reduced motility Male infertility

Catsper1,Catsper2,Catsper3,Catsper4 Sperm motility and fertilization (ion channels) Normal sperm counts, normal morphology, markedlyMale infertility reduced motility111–114 and unable to

(Cation channel, sperm associated 1–4) fertilize intact eggs b Capza3(Capping protein (actin filament)Spermatid cytoplasmic extrusion Excess cytoplasm and impaired motility Male infertility 46

muscle Z-line, alpha 3)

Dnahc1(Dynein, axonemal, heavy) chainCilia and 1 flagella function; axoneme movement Normal sperm counts, normal morphology, markedly reduced forward156 motility Male infertility and primary

ciliary dyskinesia

Izumo1(Izumo sperm–egg fusion 1) Membrane fusion Defect in sperm–egg fusion Male infertility 157

Akap4(A kinase (PRKA) anchor protein 4) Sperm tail assembly (scaffold protein) Normal sperm counts, normal morphology, markedly158 reduced motility Male infertility

Pcsk4(Proprotein convertase subtilisin/kexinAcrosome reaction and sperm–egg interaction Impaired fertilization Male subfertility 159

type 4)

Kcnu1(Slo3) (Potassium channel, subfamilyCapacitation and acrosome reactionImpaired (ion motility, abnormal sperm morphology (hairpin shape),Male impaired infertility sperm 115

U, member 1) transportation) capacitation and acrosome reaction

Adam3(Cyritestin) Cell adhesions (plasma membrane protein) Defect in sperm to bind to the zona pellucida Male infertility 160 sa ora fAndrology of Journal Asian Clgn(Calmegin) Folding and transport of integral membraneImpaired sperm–zona pellucida adhesion Male infertility 161

proteins (testis-specific chaperone)

Adam2(Fertilin-b) Cell adhesions (plasma membrane protein) Impaired sperm–egg membrane adhesion, sperm–eggMale subfertility fusion, migration162 from the uterus

into the oviduct, and binding to the egg zona pellucida

a Gene-trapped mouse models. b ENU mutagenized mouse model.

147

Table 3. Genes essential for spermiogenesis, epididymal maturation, capacitation and fertilization (adapted from Ref 8).

1.6 Role of cAMP and protein kinase A (PKA) in sperm function

As stated earlier, cAMP levels increase during epididymal maturation and capacitation (29). cAMP also regulates a group of proteins that are required for the expression of testis specific genes. These proteins include cAMP response element modulator (CREM), cAMP response element binding protein (CREB) and activating transcription factor (ATF). CRE motifs are present in the promoters of multiple testis specific genes. These include Tnp1, Prm1/2, testis-specific Ace, testis-specific isoform of lactate dehydrogenase (LDH) and others (32). CREM gene expression is regulated at the transcription level by the use of alternate promoters (33,34,35), alternative splicing

(36,37) and by the use of alternate poly (A) sites (38,39). CREM is essential for spermatid maturation and when CREM activity was abolished in knockout mice, it resulted in male infertility (40,41).

cAMP also exerts its effect by inducing PKA. Early observations were made regarding the role of cAMP mediated PKA activation in initiation of sperm motility. This suggests that PKA mediated phosphorylation of proteins could be responsible for sperm motility (42). This hypothesis is further strengthened by the fact that PKA Cα2 (the only catalytic subunit expressed in mature sperm) null males display lower sperm motility compared to wild type mice (16). If phosphorylation of serine/threonine residues by PKA can regulate sperm motility, this would certainly imply that there is a role for protein phosphatases that can restrict the action of PKA and play a role in regulating sperm motility.

1.7 Role of protein phosphatases in sperm function

The two major families of protein phosphatases are phosphoprotein sreine/threonine phosphatases (PSPs) and phosphoprotein tyrosine phosphatases (PTPs).

PSPs are broadly classified into phosphoprotein phosphatases (PPPs), metal-dependent phosphatases and aspartate-based phosphatases. Table 4 summarizes the different subfamilies of PPPs. Protein phosphates 1 (PP1), protein phosphatase 2 (PP2A) and protein phosphatase 2B (calcineurin) share the same 280 amino acid catalytic core and differ only at their N-and C-termini (42). 198 Cell Regulation

TableTable 1 4. Different subfamilies of PPPs (Ref 43).

Classification of Ser/Thr phosphatases.

Family Subfamily Genes Catalytic Regulatory Subunit composition Selected inhibitors Ref subunitsa subunits PPP PP1 (PPP1) 3 a, b, g1, g2 >50 Heterodimer and Inhibitor-1, inhibitor-2, [3] higher order microcystin (MC), okadaic acid, calyculin A (Cal A) PP2A (PPP2) 2 a, b PPP2R1–3 Heterodimer and Fostriecin, okadaic acid, [3] PPP2R5 higher order MC, Cal A, nodularin PP4 (PPP4) 11 IGBP1,PR4R1,Monomer and Fostriecin, okadaic acid, [3] (PP2A-like) PPP4R2, c-Re1 higher order MC, Cal A, nodularin PP6 (PPP6) 11 IGBP1Monomerand ?[3] (PP2A-like) higher order PP2B (PPP3) 3 3 Calcineurin B, Heterodimer and ?[3,41] AKAPs, calmodulin higher order PP5 (PPP5) 1 1 PP2A A and B Monomer and Okadaic acid, MC, [42] subunits? higher order Cal A, nodularin PP7 (PPP7) 2 2 ? Monomer ? [3] PPM PP2C 6 10 ? Monomer ? [3] FCPPP1 FCP1 is a highly 1 conserved 4 protein ? that is present Monomer in organisms such ? as yeast to [43] a Including splices. humans and in present in multiple isoforms. There are four isoforms of PP1 encoded by Figure 1). The MYPT1:PP1d dimer contains a charged the catalytic subunit is encoded by only two highly elongated catalytic cleft into which myosin light chain conserved genes. Like PP1, the isolated PP2A catalytic canthree bind. genes. This The provides catalytic a structural core explanationof all four forisoforms the subunit is highly is highly conserved. active in vitro,PP1γand1 andin vivo PP1is regulatedγ2 by biochemical observations that MYPT1 increases the formation of hetero-oligomers. By far the most abundant activityare alternatively of PP1d toward spliced MLC products while reducing from its a activitysingle gene.form These is a heterotrimer two isoforms containing differ a conservedonly in A subunit towards unrelated substrates such as glycogen synthase. and one of a large number of B subunits (see Table 1) that The remodeling of and restriction of access to the cat- serve to both target and regulate the enzyme. Formation alytic site provides a powerful paradigm for how a subset of the heterotrimer is essential for PP2A stability, since of phosphatase regulatory subunits modifies phosphatase knockdown of any one class of subunits (A, C or all of the activity. B subunits) reduces both the abundance and the stability of all the other subunits [13,14]. In addition to targeting subunits, PP-1 is regulated by several protein inhibitors, many of which are in turn reg- The high and non-specific phosphatase activity of free C ulated by phosphorylation. For example, the MYPT1: subunit is probably toxic to cells. In fact, recent studies of PP1c complex is specifically inhibited by a 21-kDa protein PP2A holoenzyme formation suggest there is significant inhibitor, CPI-17, that is activated by phosphorylation. regulation of PP2A biogenesis. Fellner et al.[15] identi- CPI-17 binds to MYPT1-bound PP1 but not to G(M)- fied a key function for a previously described PP2A bound PP1 (G(M) targets PP-1 to glycogen) [11]. This interacting protein, known as RRD1 and RRD2 in yeast, complex combinatorial control has led Bollen and cow- and PTPA (PPP2R4) in mammals [16]. Deletion of the orkers to propose a model to explain the ability of PP1 to yeast RRD proteins leads to formation of PP2A hetero- bind numerous and structurally unrelated proteins [7,12]. trimers with markedly decreased activity. Not unexpect- Although some subunits might employ unique binding edly, therefore, knockdown of the mammalian homolog sites on PP1c, most regulatory proteins bind PP1 using PTPA by RNAi causes apoptotic cell death. The most different combinations of known binding sites. Thus, likely function of PTPA appears to be facilitating inser- specific effects on PP1 could be explained by the occu- tion of required divalent cations into the phosphatase pancy of specific combinations of binding sites, which, active site, a step that may not happen until the enzyme is ultimately, have different effects on the catalytic site. in its heterotrimeric state. The association of PTPA with PP2A may be regulated by reversible methylation of the Protein phosphatase 2A: life and death PP2A catalytic subunit. PP2A may therefore remain inac- Recent studies indicate that distinct forms of PP2A tive (perhaps without its required divalent cations) until regulate development, cancer, apoptosis and circadian the holoenzyme is formed, hence preventing the less- rhythm, to name just a few of the most prominent regulated forms of the enzyme from wrecking havoc with examples. PP2A makes up 0.1% of cellular protein, yet the cell.

Current Opinion in Cell Biology 2005, 17:197–202 www.sciencedirect.com 19

their C-terminus, PP1γ2 has a unique 21-amino acid extension. PP1α, PP1β and PP1γ1 are ubiquitously expressed in all tissues whereas PP1γ2 is highly enriched in testis (42).

PP1 family of phosphatases bind to a plethora of proteins that regulate its activity and play a role in targeting it to various sub-cellular compartments.

Serine/threonine phosphatases were first discovered in protein extracts of sea urchin and bovine sperm . Western blot and enzyme activity analyses showed that the predominant serine/threonine phosphatase expressed in sperm is PP1γ2. Further analyses into the possible role of PP1γ2 revealed that it displayed differential activity in regions of the epididymis. PP1γ2 activity was higher in sperm from caput epididymis (immotile sperm) and lower in sperm from caudal epididymis (motile sperm). This suggests that

PP1γ2 could be playing an important role during the epididymal maturation of sperm

(42). To elucidate the function that PP1γ2 could be playing in sperm function Ppp1Cc gene was knocked out in mice. This resulted in mice that lacked both isoforms – PP1γ1 and PP1γ2. Interestingly, only male mice were infertile and closer analyses revealed that this was due to a spermiogenic defect and increased apoptosis of germ cells (45). Since, male mice lacked both isoforms it was not possible to determine whether lack of both isoforms or either one was responsible for this phenotype.

PP1 activity is regulated by its interacting partners and till date more than 200

PP1 interacting proteins have been identified (45). PP1γ2 is the only isoform of PP1 expressed in sperm and our lab has shown that its activity in sperm is regulated by proteins such PPP1R2 (Inhibitor-2, I2) (46,47) and PPP1R11 (Inhibitor-3, I3) (48). We

have identified other PP1γ2 binding proteins but their role in sperm function is still under investigation. These include PPP1R7 (sds22) and 14-3-3 (42).

1.7.a Role of I2 in sperm function

During the purification and characterization of heat stable inhibitors of PP1 from rabbit skeletal muscle two inhibitors were identified. One eluted of DEAE-cellulose at

0.11M NaCl and the other at 0.19M NaCl and hence called inhibitor-1 (PPP1R1, I1) and inhibitor-2 (I2) respectively. I2 is a hydrophilic and highly acidic protein and due to this it migrates anomalously on SDS-PAGE. It was also observed that I1 had to be phosphorylated to exert its inhibitory effect on PP1 and I2 inhibits PP1 when it is dephosphorylated (49). PP1 was known to be present in an inactive from due to its inhibition by I2 but this inhibition could be reversed when the inactive PP1 complex was incubated with Mg-ATP and a protein called FA. FA was later identified to be GSK-3 and phosphorylates I2 and makes PP1 active (50-52). The presence of I2-like activity was shown in heat stable sperm extracts (46,47) and this activity was thought to be that of I2 because PP1 inhibition could be reversed using glycogen synthase kinase-3 (GSK-3).

GSK-3 phosphorylates I2 at Thr-72 and once phosphorylated it does not dissociate from

PP1 (53).

Northern blot analyses of rabbit tissue revealed the presence of two transcripts of size 2.4kb and 1.6kb. The presence of these different sized messages was due to the difference in size of their 3’ UTR that was due to presence of different poly (A) sites

(54). A highly testis specific message was also observed in rabbit and rat and its size was

between 1.0 - 1.2kb (54,55). This testis-specific message was postulated to be due to the presence of an alternatively spliced isoform of I2 (55).

The presence of I2 has been shown in sperm by biochemical assays but its physical presence has never been confirmed. EST evidence and northern blot data support the presence of alternatively spliced isoform of I2 in testis and raises the question if this isoform is present in sperm and whether it plays a specific role in sperm function.

1.7.b Role of I3 in sperm function

I3 is another known heat-stable inhibitor of PP1 activity. I3 exhibits many characteristics of I2 - its inhibitory action in the dephosphorylated form (56); it is hydrophilic and is rich in acidic residues that are responsible for its anomalous migration in SDS-PAGE. It was discovered as PP1-binding protein by yeast two-hybrid assays (56).

The gene for I3 is localized on chromosome 17 within the t-complex, a naturally occurring polymorphism (57) and hence I3 is also known as tctex-5.

Northern blot analyses showed the presence of a transcript that was highly enriched in the testis that is smaller in size compared to the ubiquitously expressed message (unpublished data). EST database analyses suggest the presence of an alternatively spliced isoform of similar length.

1.7.c Role of sds22 in sperm function

In S. pombe, dis2+eencodes for a protein highly similar to the rabbit PP1 catlytic subunit (58) and its mutant form, dis2-11 causes mitotic arrest (59). A mutant of the PP1

homolog in A. nidulans causes a similar phenotype (60). sds22, a leucine-rich 22 amino acid protein, was identified in yeast to be a PP1 binding protein. It was called sds22, suppressor of dis2-mutation, and its amino acid composition (61). There have been reports that sds22 could activate (61) and inactivate PP1 (61,62).

Analyses of caudal epididymal sperm suggested that sds22 could be a possible regulator of PP1γ2 activity (63). Enzyme assay of PP1γ2 bound to sds22 revealed that it was inactive (63). Further analysis showed that in caput epididymal sperm sds22 is not bound to PP1γ2 and bound PP1γ2 is catalytically inactive (64) whereas, PP1γ2 (unbound to sds22) is inactive caudal epididymal sperm suggesting a possible role for sds22 in epididymal maturation of sperm.

Northern blot analyses of human tissue revealed that there are three transcripts coding for sds22 (65) and another study showed that six different transcripts are produced as a result of alternative polyadenylation and skipping of exon 2 (67).

1.7.d Role of 14-3-3 in sperm function

14-3-3 was identified as an abundant acidic protein in brain. The protein was named as such because of the column fraction of column chromatography during its purification and its migration position on starch gel electrophoresis. 14-3-3 is an essential protein in budding yeast and this fact is demonstrated by the fact that cells lacking 14-3-3 are non-viable (67). There are seven isoforms of 14-3-3 coded by seven different genes

(68). The exact function of this protein has not been deciphered but it is thought to play a

role in signaling pathways by altering the localization and activity of bound proteins

(68,69), similar to PP1 binding partners.

The presence of 14-3-3 in sperm was demonstrated by micro-sequencing of purified PP1γ2 fractions. 14-3-3ζ co-eluted along with phospho-PP1γ2 during purification and the interaction between the proteins was confirmed by immunoprecipitation (70). The role that 14-3-3 plays in regulating PP1 activity is still unknown.

1.8 PP1γ2 protein complexes in sperm

Analyses of sperm protein extracts using immunoprecipitation, FPLC and GST- pulldown revealed that PP1γ2 is bound to I3, sds22 and actin. PP1γ2 present in this complex was found to be inactive demonstrated by PP1 enzyme activity assay (48).

During the analyses of HPLC purified fraction by non-denaturing gel electrophoresis another protein was found to interact with PP1γ2 – RSPH1. In fact, RSPH1 was shown to bind to another PP1γ2 binding partner, 14-3-3, in sperm using in vitro (71) and in vitro

(72) analyses. These findings suggest that PP1γ2 in sperm could possibly interact with different subsets of its binding partners during different stages of sperm maturation enabling PP1γ2 to carry out its role.

1.9 Role of Rsph1 in sperm function

In mice, a novel testis-specific gene was identified from germ cell specific cDNA library and mapped to chromosome 17. It was called testis-specific gene A2 (Tsga2).

Northern blot analysis showed that the message was highly expressed in testis and a low level in ovary (74). Another group identified a testis-specific protein from testis-cDNA library that was highly expressed in pachytene spermatocytes to round spermatids and was not expressed in somatic cells. The protein was called meichroacidin because of its localization and its highly acidic nature. Using polyclonal antibodies, it was shown that this acidic protein localized to the cytoplasm but during meiosis it was associated with the metaphase chromosome and the spindles of male germ cells and hence the name meichroacidin (male meiotic metaphase chromosome-associated acidic protein) (75).

A homolog of meichroacidin was identified in carp in an attempt to identify genes involved in spermatogenesis. Northern and western blot analyses demonstrated that it was highly expressed only in the testis. This protein was shown to contain seven MORN

(membrane occupation and recognition nexus) motifs and localized to the sperm flagellum was named MSAP (MORN motif-containing sperm-specific axonemal protein). The expression of this homolog during spermatogenesis differed from meichroacidin. Immunohistochemistry analysis revealed that MSAP was expressed in late spermatids and mature spermatozoa. This suggests a possible role that it could play in sperm morphogenesis during spermiogenesis. Western blot analysis also showed that meichroacidin was not detected in rodent epididymides (75). MORN motifs, which are present in the junctophilin (JP) family, has been shown to play a role in targeting JP to the plasma membrane and for the formation of intracellular junctions (76). The presence of MORN motifs along with immunocytochemistry data showing that MSAP localization to the flagellum suggests that it could play a possible role in flagellar assembly during

spermiogenesis (75). Also, in muscle it has been shown that JP-proteins play a role in

Ca2+ channel-dependent coupling of excitation and contraction (76). The role of Ca2+ in carp sperm motility initiation has been reported (77).

Tsga2 was mapped to t-complex region in the proximal part of chromosome 17

(78). During the analysis of t-complex in mice in an attempt to identify new candidates involved in t-complex, two isoforms of TSGA2 were identified using RT-PCR and monoclonal antibodies. MORN motifs were also recognized in both isoforms of TSGA2.

Western blot analysis demonstrated that the two isoforms have a significant difference in molecular weight. The larger protein migrated at 44kDa whereas, the smaller isoform migrated to 30kDa. RT-PCR and sequence analysis showed that the smaller isoform lacked exon 2 due to alternate splicing of the larger isoform. RT-PCR and western blot data showed that the lower isoform was highly enriched in the epididymides of mice (78) suggesting a possible role for this isoform in epididymal maturation of spermatozoa.

Immunocytochemistry data demonstrated the localization of these proteins in the sperm tail and in the region of the anterior acrosome suggesting a possible role in sperm-egg interaction (78).

A human homolog of Tsga2 was identified during the analysis of sera from infertile men. Human Tsga2 was mapped to chromosome 21 and the protein contained the MORN motif like mouse TSGA2. Northern blot and western blot analyses demonstrated the presence TSGA2 in testis and other ciliated tissues.

Immunoelectronmicroscopy revealed that the protein localized to the radial spokes of the axoneme in the sperm flagellum. A suggestion to rename the protein to RSP44 (radial

spoke protein, mol. wt. 44kDa) was suggested based on its distribution and localization

(79). It was later renamed to radial spoke protein 1 homolog (Chlamydomonas).

Male mice lacking Rsph1, in which a targeting vector replaced exons 1-3 with a neomycin cassette, were infertile due to impaired spermatogenesis whereas, female mice were not affected. Caput epididymides from knockout mice rarely contained sperm, the few testicular sperm that were observed had few structural defects and the head morphology was normal. Testicular sperm from these mice were incapable of fertilization via ICSI. Electron microscopy of testis sections revealed the arrangement of mitochondria and flagellum formation were disrupted and elongated spermatids were phagocytosed by Sertoli cells. These observations suggest that RSPH1 could be involved in spermiogenesis. Immunocytochemistry and western blot data suggest that RSPH1 is present in the axoneme and ODF. Single nucleotide polymorphism (SNP) analysis of oligospermic and azoospermic human subjects revealed two SNPs that resulted in amino acid substitution in RSPH1 (81). This phenotypic defect in spermiogenesis is very similar to infertile male mice exhibiting defects in spermiogenesis when PP1γ is knocked out

(44).

PP1γ2 in testis and sperm has been to shown to interact with inhibitors of its activity such as inhibitor 2 (PPP1R2, I2) and inhibitor 3 (PPP1R11, I3) (29,42). Ppp1r11 has been mapped to chromosome 17 to the t-complex region (57); this is the same locus where Rsph1 is mapped to (73). Native PAGE analysis of FPLC purified testis extracts showed that I3 and PP1γ2 interact with RSPH1 (unpublished data). The phenotype of

Rsph1 knockout mice clearly suggests an important role in spermiogenesis and the

interaction of RSPH1 with PP1γ2 and its binding partners suggests that RSPH1 could play role in regulating spermiogenesis along with PP1γ2 and its binding partners. The presence of an epididymal isoform of RSPH1 warranted analyses the epididymal role of this particular isoform.

This led me to formulate my first aim of my dissertation. I wanted to analyze the role of the epididymal isoform of RSPH1 in the epididymal maturation of sperm and analyze the expression levels of PP1γ2 and it binding partners in Rsph1 knockout mice.

EST evidence and northern blot analyses suggest the presence of alternatively spliced isoforms of PP1 binding proteins I2, I3 and sds22. My second aim comprised of detecting the presence of these transcripts in testis, the protein in testis and sperm and to elucidate the function of these isoforms in regulating PP1γ2 activity and sperm function.

PP1γ2 is the predominant isoform of PP1 expressed in testis, the only isoform of PP1 in sperm and male mice lacking Ppp1Cc gene are infertile due to the lack of mature sperm.

During analyses of transgenic mice expressing PP1γ2 in Ppp1Cc knockout background revealed that low levels of PP1γ2 could rescue spermatogenesis but not spermiogenesis

(81). The absence/low levels of an important serine/threonine phosphatase in sperm will cause a change in phosphoproteome of sperm. Analysis of phosphoproteome of sperm from these transgenic mice could aid in the identification of possible PP1γ2 substrates, which would be phosphorylated in transgenic samples and hypophosphorylated/unphosphorylated in control sperm samples. Before analyzing the phosphoproteome, I wanted to compare the proteome of sperm from transgenic mice with sperm from Ppp1Cc +/- mice using 2D-DIGE.

To summarize, the three aims of my dissertation are:

(i) To elucidate the role of Rsph1 in spermatogenesis and sperm function.

(ii) To characterize the role of PP1 binding proteins I2, I3 and sds22 isoforms

in spermatogenesis and sperm function.

(iii) To initiate studies in order to identify possible PP1γ2 substrates.

Chapter 2

Materials and Methods

2.1 Antibody purification

The peptide against which each custom-made antibody was raised was first coupled to a SulfoLink column (Pierce) and the uncoupled sites on the beads are blocked by

L-Cysteine-HCl. Serum from immunized rabbit is passed through the column and the bound antibody is first eluted by 3M KSCN and then by 0.1M glycine (pH 2.7).

Figure 7: Schematic depicting coupling of sulfolink column with peptide against which the antibody was raised.

The KSCN and glycine eluates are dialyzed for 24 hrs in two changes 0.05% NaN3 in 1X

0 0 PBS at 4 C. The concentration is checked at A280 and the antibody was stored at -80 C.

2.2 Genotyping of Rsph1 knockout mice

Rsph1 heterozygous (+/-) mice were a kind gift from Dr. Tanaka (80). The mice were genotyped and set up for breeding with CD1 female mice to generate knockout mice.

29 30

Primer Band Genotype Primer Sequence Set Size +/+ +/- -/-

5’-CCTTGCGCAGCTGTGCTCGACGTTG-3’ Neomycin 135bp X √ √ 5’-GCCGCATTTGCATCAGCCATGATGGA-3’

5’-GAGAACGACACGGACATGGGAAAG-3’ Exon 2-3 300bp √ √ X 5’-CTTCATATCTGGATCCATCTGGA-3’

Table 5: Primers used for genotyping Rsph1 knockout mice.

2.3 Protein extract preparation

Tissue (approx. 5mg/ml) was homogenized in Hb+ (homogenization buffer) 2X for 15 seconds with a 1 min interval on ice using a homogenizer and spun down at 16,000 x g for 15 min at 4 0C. The supernatant was collected for further analysis. Sperm was collected from the caudal epididymis and vas deferns. The contents of the vas deferens were gently squeezed out using forceps into a Petri dish containing 1X PBS and transferred to a 1.5ml microcentrifuge tube using a cut pipette tip. The caudal epididymis was punctured using a needle and the contents were gently squeezed into 1X PBS. The epididymis was kept in 1X PBS for 20 min to allow the sperm to swim out. The sperm suspension was collected using a cut pipette tip and pooled with the sperm from the vas deferens. The sperm suspension was washed twice in 1X PBS at 400 x g at 4 0C and resuspended in an appropriate volume of Hb+ and sonicated. After sonication, it was spun down at 16,000 x g for 15 min at 4 0C. The supernatant was collected for further analysis.

Alternately, the washed sperm pellet was resuspended in 1% sds and boiled for 5 min and centrifuged at 10,000 x g for 10 min at 4 0C. The supernatant was collected for SDS-

PAGE analysis.

2.4 SDS-PAGE and western blot analysis

Protein extracts were boiled in Laemmli sample buffer and stored in -20 0C for

SDS-PAGE. Protein extracts were quantified using DC Assay Kit (Bio-Rad) after 10%

TCA (trichloroacetic acid) precipitation and the pellet resuspended in 0.1N NaOH. BSA

(bovine serum albumin, Sigma Aldrich) standards were subjected to the same treatment.

The assay was performed according to the manufacturer’s instructions. The protein samples were electrophoretically separated on 12% gels and transferred on to

Immobilon-P PVDF (polyvinylidene difluoride) membrane (Millipore Corp., Billerica,

MA, USA) and blocked in 5% non-fat dry milk in 1X TTBS. Following blocking, membrane was incubated with the appropriate antibody diluted in 5% non-fat dry milk in 1X TTBS overnight at 4 0C on an orbital shaker. The following day, membrane was rinsed in 1X TTBS and incubated with the appropriate secondary antibody conjugated with horseradish-peroxidase in 5% non-fat dry milk in 1X TTBS for 1hr at room temperature. The membrane was rinsed with 1X TTBS 2X for 10 min and developed using “homemade” enhanced chemiluminescence (ECL). The images were captured using a Fujifilm Darkbox LAS -3000 (Fuji).

2.5 RNA extraction and cDNA preparation

Approximately 5mg of tissue was homogenized in 1ml of TRI reagent (Sigma

Aldrich) and total RNA was extracted using manufacturer’s instructions. cDNA was prepared from 1 µg of total RNA using QuantiTect Reverse Transcription Kit (Qiagen).

Spermatid cDNA library was purchased from ATCC. The transcripts for testis specific isoforms were isolated using RT-PCR. The PCR products were subcloned into pGEM-T

Easy cloning vector (Promega) and sent to the Genomics Core at Lerner Research

Institute for sequencing.

Figure 8: Map of pGEM – T Easy vector used for cloning of testicular isoform messages.

2.6 Northern blot analysis

Total RNA (20 or 25µg) of each sample was mixed with a 17µl reaction mixture containing 2µl of 10x MOPS (3-N-morpholinopropanesulfonic acid) buffer, 4µl of

HCHO (37% solution; Amresco, Solon, OH), 10µl of deionized formamide (Amresco), and 1µl of ethidium bromide (200 µg/ml). The mixture was mixed by brief vortexing and then heated to 85 0C for 10min and then cooled on ice for 10min. 2µl of 10x gel loading dye (50% glycerol, 10mM EDTA (ethylenediaminetetraacetic acid) pH 8.0, 0.25% w/v bromophenol blue, and 0.25% w/v xylene cyanol FF) was added to each sample and mixed before loading. Samples were electrophorectically separated on 1.5% agarose/MOPS (1X)/ HCHO (1.8X of 37% solution) gel at 70 volts for 3.5 to 4 hrs.

Following separation, RNA was immobilized on positively charged Hybond-XL nylon membrane (GE Healthcare, Piscataway, NJ) by capillary transfer in 10xSSC. Transfer was allowed for 16-18hrs overnight, followed by baking at 85 0C for 2 hrs in vacuum oven. Membrane was prehybridized in 10ml of modified church buffer (1mM EDTA pH

0 8.0, 0.5M NaHPO4 pH7.2, and 5%SDS) in a sealed nylon bag in a water bath at 65 C for

1 hr. Probe was labeled by random labeling using 32P-dCTP using Rediprime nicktranslation kit (GE Healthcare, Piscataway, NJ) following manufacturer’s protocol.

Probe was purified using Illustra NICKTM columns (GE Healthcare, Piscataway, NJ) following manufacturer’s protocol. The purified radiolabeled probe was diluted in 10ml of modified church buffer and added to the bolt in a sealed nylon bag. The blot was incubated overnight at 65 0C in a water bath at 50rpm. After hybridization, the blot was washed 2X for 5 min in 200ml of wash buffer I (1% SSC, 0.1% sds, high stringency

wash) followed by 1X for 5 min in wash buffer II (0.1% SSC, 0.1% sds, low stringency wash) at 45 0C. After washing, the membrane was wrapped in saran wrap and exposed to phosphor-imager screen (Molecular Dynamics) and developed using Typhoon scanner

(GE Healthcare, Piscataway, NJ) or the membrane was exposed to X-ray film and developed.

2.7 Protein purification from testis and sperm extracts.

Testis and epididymal protein extracts were purified using AKTA-FPLC (GE

Healthcare). Epididymal protein extracts were purified using HiTrap DEAE FF (GE

Healthcare) followed by HiTrap Q FF (GE Healthcare) for the purification of P30 isoform of Rsph1 using a 1M NaCl gradient spread over 7 ml. Testis extracts were purified using Capto-DEAE FF (GE Healthcare) for the purification of I2, I3 and sds22 using a 1M NaCl gradient spread over 7ml. Sperm protein extracts were purified using

DEAE-Sepharose FF beads (GE Healthcare). Sperm protein extracts were added to beads that were washed with 1ml 20mM Tris pH7.0 3X at 4000 rpm for 1 min at 4 0C. The beads-extract suspension was incubated on a rotator at 4 0C for 2 hrs. The suspension was spun down and washed with 20mM Tris, 50mM NaCl, pH 7.0 and then eluted with

20mM Tris, 200mM NaCl, pH 7.0. The different fractions were analyzed using SDS-

PAGE and immuno-blotting using specific antibodies to ascertain the fractions in which the proteins of interest were present. The fractions of interest were subjected to SDS-

PAGE and then coomassie stained and sent to the Proteomics Core at Lerner Research

Institute for sequencing analysis.

2.8 Immunoprecipitation

500 µL of testis extracts was incubated with 2.5 µg of anti-InA, anti IQUB and whole rabbit IgG (Jackson Immunoresearch) for 90 min on a rotator at 4 0C. 30 µl of protein G-Sepharose beads (GE Healthcare) slurry was washed with 1X PBS 3X at 4000 rpm for 1 min at 4 0C. The antibody-extract mixture was added to the beads and incubated on a rotator for 1 hr at 4 0C. After an hour, the beads were spun down and the flow through was collected. The beads were washed 3X with 1X TTBS followed by another wash in Hb+. The beads were finally resuspended in 30 µl of Hb+, boiled with

Laemmli sample buffer and analyzed by western blot.

2.9 Immunohistochemistry

Epididymis was surgically removed and immediately fixed in freshly prepared 4% paraformaldehyde in 1x PBS for 12-16 hrs at 4 0C. Fixed tissues were dehydrated by continuous treatment in increasing concentrations of ethanol (70%, 95%, and 100%) for

45mins each and then permeabilized in CitriSolv (Fischer Scientific) for 30 min. Tissues were then embedded in paraffin and 8µm thick sections were cut using a microtome

(Leica) and transferred to poly L-lysine coated slides. Sections were de-paraffinized in

Citrisolv and rehydrated by continuous treatment in decreasing concentrations of ethanol

(100%, 95%, 80% and 70%) as previously reported (39,50). De-paraffinized, rehydrated sections were boiled in citrate buffer (10mM Citric acid, 0.05% Tween20, pH 6.0) for antigen retrieval followed by incubation in blocking media (10% goat serum, 5% BSA in

1x PBS) for 1hr at room temperature. Sections were incubated with aforementioned anti-

InA and anti-R2K primary antibodies at 1:250 dilutions (in blocking solution) at 4 0C overnight followed by three brief washes in 1x PBS. Negative control sections were incubated in blocking solution instead of primary antibody. Sections were subsequently incubated with corresponding Cy3-conjugated secondary antibody (Jackson

Immunoresearch) for 1hr at room temperature. Finally sections were washed several times for 10 min each time in 1X PBS and mounted with Vectashield (Vector

Laboratories, Burlingame, CA, USA) mounting media, then examined using a

Fluoview 500 confocal fluorescence Microscope (Olympus, Melville, NY, USA).

Chapter 3

Results

3.1.

Aim 1: To elucidate the role of Rsph1 in spermatogenesis and sperm function.

Rationale:

Our interest in examining the role of RSPH1 in spermatogenesis and sperm function is due to:

1) Rsph1 is localized within the t-complex region of chromosome 17 in mouse, a region that is important for male fertility (57,73),

2) it is expressed in two alternatively spliced isoforms – P44 and P30 that are expressed in the testis and that epididymis respectively (78).

Targeted deletion of Ppp1Cc leads to male infertility due to defects in spermiogenesis (44). The phenotype exhibited by these knockout mice is very similar to the phenotype of mice lacking Rsph1 (44,80). Male mice lacking Rsph1 are also infertile due to defects in spermiogenesis (80). These observations led us to analyze the role that

RSPH1 has in PP1γ2 signaling and in spermatogenesis. Analysis of FPLC purified testis by native-PAGE demonstrated that RSPH1 co-migrates with PP1γ2 and its binding partners I3 and sds22. Evidence of interaction between RSPH1 and I3 was further strengthened by immunoprecipitation of FPLC purified testis extracts with anti-I3 that demonstrated that RSPH1 interacts with I3. In vivo and in vitro experiments demonstrated that RSPH1 interacts with another PP1γ2 binding protein -14-3-3 (71,72).

37 38

Our hypothesis was that P40 was essential for spermatogenesis whereas P30 was required for epididymal sperm maturation. We also proposed that RSPH1 could be a regulator of

PP1γ2.

3.1.1 Strategy to determine the role of P30 in epididymal sperm maturation.

Rsph1 -/- mice lack both isoforms of RSPH1. Transgenic mice expressing P44 and not P30 will enable us to study the role of P30 in epididymal sperm maturation.

These mice will lack P30 and analyzing the phenotype will help us elucidate the role of

P30 in epididymal maturation.

Figure 9: Strategy to elucidate the role of P30 isoform in sperm maturation. The figure above depicts the differential expression of the two isoforms of RSPH1. P44 is preferentially expressed in the testis (green) and P30 in the corpus and caudal epididymydes (green). Using the Rsph1 -/- mice, which lack both isoforms, P44 rescue mice (blue) will be generated and this enable us to study the role of P30 in epididymal sperm maturation.

3.1.2 Sequence of RSPH1 depicting the epitopes used for antibody production.

RSPH1 is expressed as two isoforms – P44 and P30. They were proposed to be formed as a result of alternate splicing (78). The smaller, epididymal isoform P30 proposed to lack exon 2. The N-terminus antibody was raised against an epitope that is coded by exon 2 and will only recognize the larger isoform – P44. Whereas, the C- terminus will recognize both isoforms of RSPH1. These antibodies were a gift from Dr.

Pilder.

Figure 10: The region in red is exon 2, spliced out in the epididymal-isoform (P30) but present in the testis-form (P44) of RSPH1. The membrane occupation and recognition nexus (MORN) motifs are shown on a gray background and the acid-rich regions (ARRs) are underlined. Residues in bold typeface are the N- and C-terminal sequences used for antibody production. Note that the N-terminus antibody will recognize only the testis isoform whereas the C-terminus antibody will recognize both isoforms.

3.1.3 Presence of RSPH1 isoforms in testis and sperm.

To determine the presence of Rsph1 isoforms, testis and sperm extracts were separated by SDS-PAGE followed by Western blot analysis with N-terminus or with C-

terminus antibody. Testis and spermatozoa contain P44. Sperm extracts reveal a protein band at 30-KDa with the C-terminus but not with the N-terminus antibody.

Figure 11: N-terminus antibody (left panel) recognizes the P44 isoform of RSPH1 in testis and sperm whereas, the C-terminus antibody recognizes both isoforms in sperm and only P44 in testis.

As previously mentioned, the N-terminus antibody will recognize only the testicular isoform of Rsph1 – P44 whereas, the C-terminus antibody will recognize both

P44 and P30. The additional observation that P44 was present only in the testis and both isoforms are present in sperm lead us to our next question – whether P30 was a proteolytically processed from P44 or do sperm acquire this isoform during their maturation in the epididymis. Before addressing these questions we first set out to determine if P30 was expressed in all regions of the epididymis or if it was expressed in a region-specific manner in the epididymis.

3.1.4 Region-specific expression of P30 in the epididymis.

As noted earlier P44 isoform of Rsph1 was shown to be expressed in the testis and P30 in the epididymis(78). The specific region of the epididymis where it was expressed was not determined. To determine the regions of the epididymis where P30 is expressed, protein extracts from three regions of mouse epididymis - caput, corpus and caudal were made and analyzed by western blotting using the C-terminus RSPH1 antibody. This shows the expression of P30 in the corpus and caudal regions but not the caput regions of the epididymis. To determine if sperm acquire P30 during their maturation in the epididymis or if P30 was a result of proteolysis of P44, caudal epididymis from Ppp1Cc -/- mice were analyzed by western blot. These mice lack sperm in their epididymis, determintation of whether P30 was present in the epididymis of the null mice will show us whether protein is already present in sperm or whether it derived from the epididymal secrtions. Western blot analysis reveals that Ppp1Cc -/- mice indeed express P30 in the caudal epididymis (A, Center panel). This suggests that sperm acquire

P30 in the epididymis from the luminal fluid. These conclusions were further confirmed by immunohisotchemical analyses of Ppp1Cc -/- epididymis that demonstrates that P30 is present in the corpus epididymal epithelium (B).

Figure 12:A. 1.Western blot analysis of caput (Cap), corpus (Corp) and caudal (Cd) regions of the +/+ epididymis with the C-terminus antibody. P30 is present in corpus and caudal regions but not in the caput region of the epididymis. 2 and 3.Western blot analysis of testis (Ts) and cauda epididymis (Cd) proteins from wild type (+/+) and Ppp1cc null (-/-) mice (the latter contains few spermatozoa) showing that P30 is made in the epididymis; (Right) B. Immunohistochemical analysis of caput and corpus sections from Ppp1cc -/- and +/+ epididymides probed either with or without the C-terminus anti- RSPH1 antibody. Note the adluminal location of P30 in the -/- corpus and its adluminal and luminal location in the +/+ corpus.

The results above show that P30 is expressed in a region-specific manner in corpus and caudal epididymis and not in the caput. This lead us to hypothesize that P30 could be playing a role in the epididymal maturation of sperm. Also, we confirmed that

P30 was not proteolytically processed from P44 and was in fact secreted by the epididymal epithelial cells into the epididymal luminal fluid where sperm acquire it.

3.1.5 New RSPH1 antibody raised against a third epitope.

The determination that P30 was secreted into epididymal luminal fluid by epididymal epithelial cells lead us next to analyze its biological role in the epididymis.

We decided to use bovine epididymal luminal fluid because it easier to obtain the fluid free from spermatozoa and in substantially larger amount than from mice. The two antibodies that we had in hand do not recognize bovine RSPH1. In order to conduct studies with bovine epididymal fluid we decided to raise a new antibody against a third epitope that would recognize both bovine and murine homologs of RSPH1. This antibody should also recognize both P44 and P30

1 MSDLGSEELEEEGENDLGEYEGERNEVGERHGHGKARLPNGDTYEGSYEFGKRHGQGTYK 61 FKNGARYTGDYVKNKKHGQGTFIYPDGSRYEGEWADDQRHGQGVYYYVNNDTYTGEWFNH 121 QRHGQGTYLYAETGSKYVGTWVHGQQEGAAELIHLNHRYQGKFMNKNPVGPGKYVFDIGC 181 EQHGEYRLTDTERGEEEEEEETLVNIVPKWKALNITELALWTPTLSEEQPPPEGQGQEEP 241 QGLTGVGDPSEDIQAEGFEGELEPRGADEDVDTFRQESQENSYDIDQGNLNFDEEPSDLQ 301 D

Figure 13: Protein sequence of mouse Rsph1 depicting the homologous sequence (in blue) against which the bovine antibody was raised. This antibody was made because the mouse C-terminus antibody does not recognize bull RSPH1.

Figure 14: Validation of new RSPH1 antibody. A.The new Rsph1 antibody detects the P44 isoform in mouse testis (T) and caudal sperm (S). Testis extracts were prepared in Hb+ and sperm extracts were prepared in 1% sds. B. The new RSPH1 antibody detects the P44 isoform in bull testis (T) and caudal sperm (S). Testis extracts were prepared in RIPA+ and sperm extracts were prepared in 1% sds.

The new RSPH1 antibody detects P44 in both mouse and bull testis but surprisingly did not show the presence of P30 in either mouse or bull sperm. This raised the possibility that P30 could be a cross-reacting protein that the C-terminus RSPH1 antibody reacts against and that P30 may not be an alternately spliced isoform of RSPH1.

3.1.6 P30 is a cross-reacting protein.

To determine whether P30 was a cross-reacting protein we compared extracts from testis and epididymis from Rsph1 -/- and wild-type mice with the N-terminus, C- terminus and the new antibody. Rsph1 null mice should lack the P44 but contain P30.

Figure 15: P30 is present in Rsph1 -/- mice. The presence of P30 in Rsph1 -/- epididymis was demonstrated by western blot analysis of Rsph1 -/- and wild-type testes and epididymis (A,right). The inability of the new RSPH1 antibody to recognize P30 was reconfirmed (B). This confirms that P30 could be a cross-reacting protein. (T- mouse testes, E- mouse epididymis).

This was indeed the case. The western above strongly suggests that P30 was a cross-reacting protein that was recognized by the C-terminus RSPH1 antibody. Due to this surprising discovery that P30 was not an isoform of RSPH1 as previously reported

(78) our initial strategy to elucidate its role in epididymal maturation and sperm function

was no longer valid. We wanted to conclusively determine the identity of P30 by protein microsequencing.

3.1.7 Identification of P30.

To determine the identity of P30, protein extracts from whole epididymis were serially purified by FPLC using DEAE and Mono S and analyzed by Western blot using

C-terminus RSPH1 antibody (Figures 16 and 17). This fraction was then separated by

SDS-PAGE and the protein band around 30kDa was microsequenced (Figure 18).

55 43

34 P30 26

Figure 16: DEAE purification of Ppp1Cc +/- epididymis protein extracts. Epididymal protein extracts was prepared in Hb+ and purified using DEAE. The wash fraction was dialyzed in 20mM Tris (pH 8.0) and further purified.

Figure 17: Mono S purification of “Wash” fraction. The “wash” fraction from DEAE purification (Figure 16) was further purified using Mono S. P30 was present in the flow through (FT) fraction.

Report 1638 Vijay

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250

150

100

50 40 band of interest band of interest 30

Figure 18: SDS-PAGEMouse of FT fraction from Mono S purification with the band for microsequencing for the identification of P30.

1 MALMLVLFFLAAVLPPSLLQDSSQENRLEKLSTTKMSVPEEIVSKHNQLR 50 RMVSPSGSDLLKMEWNYDAQVNAQQWADKCTFSHSPIELRTTNLRCGENS 100 FMSSYLASWSSAIQGWYNEYKDLTYDVGPKQPDSVVGHYTQVVWNSTFQV 150 ACGVAECPKNPLRYYYVCHYCPVGNYQGRLYTPYTAGEPCASCPDHCEDG 200 LCTNSCGHEDKYTNCKYLKKMLSCEHELLKKGCKATCLCEGKIH

Figure 19: Amino acid sequence of CRISP1 with peptides identified during microsequencing in bold.

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Figure 20: Microsequencing result identified P30 as CRISP1.

Microsequencing results of the P30 band from SDS-PAGE revealed a single protein. This protein was identified to be CRISP1. Analysis of CRISP1 amino acid sequence revealed multiple non-specific epitopes that the C-terminus RSPH1 could possibly recognize. However, how previous reports concluded that this was alternatively spliced form of RSPH1 lacking exon 2 remains a mystery.

3.1.8 Co-elution of Rsph1 with PP1γ2 and its binding partners – I3, sds22 and 14-3-

We next focused our studies to determine if RSPH1 had role in regulating PP1γ2.

We therefore investigated whether RSPH1 interacts with PP1γ2 and its binding partners,

I3, sds22 and 14-3-3. Testis protein extracts were purified by FPLC using DEAE demonstrated co-elution of RSPH1, PP1γ2, I3 and sds22.

Figure 21: Co-elution of RSPH1, PP1γ2 and PP1γ2 binding partners – I3, sds22 and 14- 3-3 in DEAE purified testis protein extracts.

Co-elution of RSPH1 and PP1γ2 and its binding partners in DEAE purified FPLC extracts suggests that RSPH1 could be existing as a complex with these proteins and thus potentially play a role in regulating PP1γ2.

3.1.9 Comparison of expression levels of PP1γ2 and its binding partners – I3, sds22 and 14-3-3 between wild-type and Rsph1 -/- mice.

Next we compared the expression levels of these proteins in Rsph1 -/- and wild- type litter-mate. Testis and epididymis protein extracts were made from Rsph1 -/- and wild-type littermate. 25µg of each sample was separated by using SDS-PAGE and western blot was carried out using anti-N-terminus Rsph1, anti-PP1γ2, anti-I3 antibodies.

This was done in order to compare expression levels of these proteins between Rsph1 -/- and wildtype mice. Actin was used as a loading control.

I3 and sds22 levels in in sds22I3 and levels

– testis and epididymis when epididymis and testis

- / -

Rsph1 2 binding partners partners 2 binding γ n was used as loading control. n was used as loading 2 and PP12 and γ 2, I3 and sds22 levels in in sds222, I3 and levels γ Comparison ofComparison PP1

: and wildtype testis and epididymis protein extracts using western blot. There is There blot. using western extracts protein epididymis and testis wildtype and

- / -

22 Figure Rsph1 PP1no difference in Acti littermate. wildtype to compared

As expected Rsph1 was not detected in the Rsph1-/-. No differences were observed in the levels of PP1γ2 and its binding partners in testis of Rsph1 -/- compared to wild-type mice.

3.1.10 Evidence that RSPH1 interacts with PP1γ2 and its binding partners PPP1R11

(I3) and PPP1R7 (sds22).

Native PAGE of Superose 6 purified testis protein lysate followed by western blot demonstrates the co-migration of RSPH1, PP1γ2, I3 and sds22 suggesting that these proteins could possibly interact in vivo.

Figure 23: Fractions of testis proteins purified by Superose 6 column chromatography were separated by native PAGE followed by western blot analysis. Quadruplicated blot strips probed with either anti-RSPH1, anti-PPP1R11, anti-PP1γ2, or anti-Sds22

antibodies, as indicated, demonstrate that RSPH1, PPP1R11, PP1γ2, and sds22 co- migrate.

3.1.11 Immunoprecipitation indicating I3 interacts with RSPH1.

Fractions of mouse testis protein extracts purified by size-exclusion column chromatography after a series of anion-exchange columns were incubated with anti-

PPP1R11 or pre-immune serum immobilized on G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for RSPH1.

Figure 24: Immunoprecipitation of testis protein extracts separated by size-exclusion chromatography with I3 antibody and probed with anti-N-terminus RSPH1 antibody indicating RSPH1 interacts with I3.

Possible interaction between PP1γ2, sds22 and RSPH1 was observed when testis extracts were enriched by anion-exchange and size-exclusion chromatography followed by native

PAGE analysis (Figure 23). Interaction between Rsph1 and I3 was confirmed by immunoprecipitation of purified and enriched testis extracts (Figure 24). While enrichment by purification showed these proteins complexed to RSPH1 immunoprecipitation and pull-down experiments with testis extracts were inconclusive in determining whether RSPH1 formed a complex with PP1g2 or it binding partners.

3.1.12 Interaction between RSPH1 and CMUB 116.

During the course our studies, it was reported in Ciona intestinalis cilia that a MORN motif containing radial spoke protein (ortholog of RSPH1) binds to CMUB116 (89), a protein that is highly expressed in testis (Figure 28). We therefore wanted to see if mouse

RSPH1 interacted with CMUB116 as in Ciona intesinalis. We generated antibodies that capable of recognizing CMUB116 in mice (Figures 26 and 27). Using these antibodies, used co-immunoprecipitation with mouse testis extracts to ascertain their interaction

(Figure 29).

Figure 25: Mouse CMUB116 (IQUB) depicting the three epitopes (red) against which the anti-CMUB116 antibody was raised.

Figure 26: Validation of Anti-CMUB116 antibody. Mouse testis (T) and sperm (S) protein extracts were used to characterize this antibody. CMUB116 was identified approx. at 130kDa.

Figure 27: Approximate expression pattern of CMUB116 inferred from EST sources demonstrating maximum expression in testis. (Source:NCBI)

CMUB116 is highly expressed in testis as shown in mouse EST database above.

Figure 28: Co-immunoprecipitation demonstrating the interaction between RSPH1 and CMUB116 in mouse testis protein extracts. Rabbit IgG was used as control.

Co-immunoprecipitation of mouse testis protein extracts shows that RSPH1 and

CMUB116 may form a complex.

Summary:

RSPH1 was proposed to be expressed as two alternatively spilced products – a 44 kDa testicular isoform (P44) and a 30kDa epididymal isoform (P30). Using isoform specific antibodies we were able to demonstrate that P30 was expressed in the corpus and caudal regions of the epididymis that suggested that it could play a role in the epididymal maturation of sperm. The presence of P30 in the corpus and caudal epididymis of

Ppp1Cc -/- mice (do not contain sperm in epididymis) suggests that sperm acquire P30 in the epididymis. This suggested that P30 is secreted by the epididymal epithelium in to the epididymal luminal fluid and is taken up by sperm from the epididymal fluid.

To analyze the role of P30 in sperm maturation and function we proposed an elegant but failed strategy Rsph1 -/- mice and generating transgenic mice expressing P44 and not

P30. Also, to analyze the luminal fluid we generated new RSPH1 antibodies that would recognize both isoforms of RSPH1 in mice and bull. During the characterization of this antibody we observed that P30 was not related to RSPH1 but indeed was the epididymal protein CRISP1. This was confirmed by protein purification and microsequencing.

Rsph1-/- and Ppp1Cc -/- mice display phenotypic defects in spermiogenesis. This observation and the observation that RSPH1 interacts with Pp1γ2 binding partners – I3, sds22 and 14-3-3 suggested that it could play role in Pp1γ2 regulation. Keeping this in mind, we compared the levels of expression of Pp1γ2 and its binding partners – I3 and sds22. The expression levels of Pp1γ2, I3 and sds22 were unaltered. We also have shown that RSPH1 interacts with another testis-specific protein CMUB116 by co- immunoprecipitation.

3.2

AIM 2 - To characterize the role of PP1 binding proteins I2, I3 and sds22 isoforms in spermatogenesis and sperm function.

Rationale:

The proteins I2, I3 and sds22 have been identified as binding partners of PP1γ2 in testis and sperm (43). The presence of I2 was determined indirectly by an enzyme assay however direct evidence for the presence of I2 in testis and sperm is lacking (46,47). The other two proteins I3 and sds22 were identified by western blot and pull-down assays using recombinant PP1γ2 (48). Examination of EST databases and recent annotation for the genes, Ppp1r2 (I2), Ppp1r11 (I3) and Ppp1r7 (sds22), in the mouse genome suggest the existence of alternatively spliced isoforms for these PP1γ2 binding proteins. There are previous reports for the presence of a unique transcript coding for a testis isoform of sds22 in rat (65) and the presence of alternate transcripts, in testis, for I2 (54,55). Aside from the prediction in the genome database, the existence of an isoform I3 has not yet been reported. How these transcripts arise, the expression patterns for these isoforms in testis and whether protein products are translated from these transcripts are not known.

Aim two of my dissertation is the identification and characterization of the transcripts for the isoforms of these proteins, determining whether they are predominant in testis and determining whether protein products corresponding to these transcripts

exist. We also planned to determine the expression of the transcripts and proteins in postnatal developing testis.

3.2.1 Validation of the antibodies for I2, I3 and sds22.

PP1γ2 binding proteins are ubiquitously expressed. First, to validate the specificity of our antibodies we determined if the antibodies that we had generated reacted against the three proteins in brain, heart, lung, kidney and testis extracts. The extracts were separated by SDS-PAGE and analyzed by western blotting. Anti- I2, I3 and sds22 antibodies made in our laboratory were used. Heat-stable protein extracts were also prepared and analyzed by western blotting for the presence of I2 and I3. Figure 29 shows that these three PP1γ2 binding proteins are ubiquitous and the antibodies we had generated are able to identify these proteins.

Figure 29: A: Western blot analysis of mouse protein lysates from various tissues and probed with C-terminus I2, I3 peptide 2 and sds22 peptide 2 antibodies demonstrating ubiquitous expression of I2, I3 and sds22. B: Heat stable protein lysate analyzed by western blot and probed with C-terminus I2 and I3 peptide 2 demonstrating the same.

It should be noted that, using the new C-terminus antibody we had generated (see below), this is the first time we were able to detect I2, especially in testis.

3.2.2 Validation of new C-terminus I2 antibody and identification of I2 in mouse testis and sperm.

Figure 30: Amino acid sequence of PPP1R2 (I2) depicting the epitopes against which antibodies were raised.

Figure 31: Validation of new C-terminus I2 antibody. The new antibody was tested against recombinant His-tagged I2 (rI2) and mouse testis extracts (T).

The new C-terminus I2 antibody recognizes I2 at the expected position in mouse testis extracts. His-tagged I2 migrates at a higher position due to the presence of a linker region in the recombinant protein. The anomalous migration of I2 at 35kDa when its calculated molecular weight is 23kDa, is documented.

Figure 32: Presence of I2 in mouse testis (T) and sperm (S).

Our laboratory had previously reported the presence of I2-like activity in bovine sperm using a biochemical assay (46,47) but had not confirmed the presence of the protein. Data in Figure 32 show for the first time identification of I2 in testis and sperm.

3.2.3 Presence of a unique message for I2.

A unique message for I2 in testis was observed (0.9 Kb in testis compared to

1.4kb in other tissues including testis) in early reports when the role of I2 in PP1 regulation was analyzed (54,55). It was however not known how this testis message was different from that seen in other tissues.

The EST database and recent gene annotation of Ppp1r2 gene predict that an alternatively spliced isoform for I2 could exist (Figure 33). This alternatively spliced isoform is predicted to arise due to retention of the intron between exons 5 and 6 resulting in a unique C-terminus (Figure 34). Splicing of this intron results in a message that codes for the ubiquitously expressed I2. The two proteins, the somatic form and testis form, would be identical in all respects except for the 14 C-terminal amino acid residues. The somatic form is 206 aa in length (as previously characterized [50-53]) whereas the testis isoform is 195 aa in length. Based on EST data this isoform was expected to be predominant in testis. Indeed Northern blot analysis of total RNA from mouse testis detects a 0.9 kb message presumably corresponding to the alternate mRNA formed due to intron retention as described above (Figures 33 and 35).

Ubiquitously expressed I2

Unique isoform of I2

Figure 33: Schematic representing the exon-intron arrangement and alternate splicing of Ppp1r2. The exons and introns are not drawn to scale.

Figure 34: Predicted amino acid sequence of the unique isoform of I2 based on gene annotation from Ensembl compared to the regular isoform of I2. The two isoforms differ in the C-terminus of their amino acid sequence.

Total Testis RNA

Figure 35: Northern blot analysis of total RNA from Regular I2 mouse testis showing the presence of two isoforms 1.4 kb of I2 when probed with full-length 0.9kb I2 cDNA probe. This probe will detect both forms. The higher 1.4kb band corresponds to the ubiquitously 0.9 kb I2 expressed isoform of I2 message and the lower 0.9kb band corresponds to the unique I2 message.

3.2.4 The 0.9kb message for I2 is present only in testis.

To determine if this 0.9kb message for I2 is ubiquitous or testis specific, Northern blot analysis of total RNA from mouse tissue was performed. The 1.4kb message is expressed at basal levels in all tissues that were analyzed, whereas the 0.9kb I2 message is highly expressed in testis (Figure 36).

Figure 36: Total RNA (25µg) tissue blot demonstrating testis-specific expression of 0.9kb message of I2 when probed with cDNA of testicular isoform of I2.

The 0.9kb message present in testis will be referred as the testis I2 -isoform. To further confirm the presence and to determine the nucleotide sequence of this message, spermatid cDNA library and testis cDNA were subjected to PCR using specific primers that were designed to detect the alternate 3’end of the tesitis isoform.

Figure 37: Agarose gel electrophoresis of the alternate isoform of I2 PCR product with a unique 3’UTR.

Figure 38: Confirmation of presence of unique testicular isoform of I2 by RT-PCR using specific primers (arrow-heads).

An approximately 0.9 kb band was observed when the PCR product was analyzed by gel electrophoresis (Figure 37). Sequencing of the PCR product revealed that an alternate transcript for I2 was indeed present in testis. This mRNA with a unique 3’UTR, different from the ubiquitously expressed I2 mRNA, results from the retention of the

terminal intron as shown in Figure 33. Our next task was to determine if the protein corresponding to this testis mRNA was present.

3.2.5 Identification of testicular isoform of I2 in mouse testis and sperm.

Since, the testicular isoform of I2 differs from the somatic isoform only by three amino acids (Figure 34), isoform-specific antibodies could not be generated. The antibodies that we had in hand detect both or only the somatic isoform of I2. In order to identify, at the protein level, the testicular isoform of I2, heat-stable mouse testis and sperm extracts were purified by FPLC using DEAE Sepharose. The column fractions were separated on SDS-PAGE and the bands where the I2 was expected to migrate were excised and microsequenced.

LC-MS was carried out in order to identify the isoforms of I2 from testis and sperm in the bands indicated in Figure 40. In the initial survey analysis, peptides corresponding to I2 were identified. The peptides that were identified were not at the C- terminus and hence, it was not possible to determine whether these peptides correspond to the ubiquitously expressed isoform or the testis isoform of I2. To overcome this predicament, selective reaction monitoring reaction (SRM) was carried out. SRM is a highly sensitive and reproducible technique that is used to identify peptides present in low abundance and quantitate these peptides. It is a form of targeted analysis by which we can identify specific peptides in the sample. SRM had to be used in the identification of I2 to overcome the limitation of data dependent scans, which make use of previously

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Figure 40: SRM analysis of DEAE purified heat-stable mouse testis and sperm protein lysate indicating the relative abundance of I2 isoforms in the three bands (Figure 38) that were excised. Isoform 1- ubiquitously expressed isoform of I2 and isoform 2 – testis isoform of I2.

In order to analyze the data in a more sensitive manner a selective reaction monitoring (SRM) was carried out. These SRM experiments involve the analysis of specific m/z ratios over the entire course of the LC experiment. To ensure successful injection and analysis it is important to usePage targeted 16 control peptides. These control peptides need to be identified in all the samples being analyzed to ensure that the sample were injected and analyzed successfully. The control peptides also aid in calculating relative abundance of the specific targeted peptides in samples being analyzed (Figure

40).

Both isoforms of I2 were identified from heat-stable testis and sperm protein extracts purified using DEAE (Figures 39 and 40). The C-termini of both isoforms were identified. This was the first time that not only the ubiquitous I2 but also a new isoform of I2 were identified in both testis and sperm. It should be emphasized that the relative abundance shown in Figure 40 is the abundance of the proteins in the column fraction

(the bands excised from the gel) and not the abundance of the proteins in testis and spermatozoa.

3.2.6 Evidence for the presence of an isoform of I3 in testis.

A recent gene annotation for I3 in the Ensembl mouse genome database, predicts two transcripts for I3. The transcripts vary at their 5’UTRs - the alternate transcript is produced due to the presence of an alternate transcription start site (Figure 41). This results in the production of a unique transcript that has exon 1b instead of exon 1a

(Figures 41 and 42) and will result in the alternate isoform having a unique N-terminus.

Figure 41: Schematic representation of mouse chromosome 17 depicting the presence of two transcription start sites (green arrows) resulting in two isoforms of I3.

Figure 42: Schematic representing the exon-intron arrangement and alternate splicing of Ppp1r11. The exons and introns are not drawn to scale.

Figure 43: Predicted amino acid sequence of the unique isoform of I3 based on gene annotation from Ensembl compared to the amino acid sequence of ubiquitously expressed I3. The two isoforms differ in the N-terminus of their amino acid sequence.

Due to the presence of an alternate transcription start site and the use of exon 1b, this alternate transcript would code for a protein that differs in its N-terminus from the I3 protein coded by the 1.5kb ubiquitously expressed message. The ubiquitously expressed

isoform of I3 is comprised of 131 aa whereas, the alternate isoform is translated into a protein 120 aa in length (Figure 43). The proteins are identical except for the N-terminus amino acids shown in the yellow box of Figure 43.

3.2.7 The unique 0.6kb message of I3 is highly enriched in testis.

Northern blot analysis of total RNA from various mouse tissues was performed in order to determine where the alternate isoform is expressed. The 1.5kb message is expressed in all tissues while the 0.6kb I3 message is predominant in testis but expressed at basal levels in other tissues as well (Figure 44).

Figure 44: Total RNA (25µg) tissue blot demonstrating testis-specific expression of 0.6kb message of I3.

This isoform of I3, henceforth, will be referred to as the testis I3. To determine the identity of this message RT-PCR of testis RNA was performed using specific oligonucleotide primers.

Figure 45: Agarose gel electrophoresis of the alternate isoform of I3 with a unique 5’UTR.

Figure 46: Confirmation of presence of unique testicular isoform of I3 by RT-PCR using specific primers (arrow-heads).

An approximately 0.6kb band was observed when the PCR product was analyzed by gel electrophoresis (Figure 45). The PCR product was purified from the gel, subcloned and sequenced. Sequencing of the PCR product showed that the the unique isoform of I3 was indeed due to the 5’ end arising from the alternate transcription start site as described above (Figures 41 and 42 ). Our next goal was to identify the protein arising from this message for I3 in testis.The antibodies we had in hand was not capable

of reacting against the testis isoform. We therefore generated an isoform-specific antibody against the predicted amino acid sequence of the testiscular isoform and yet another antibody that should recognize both I3 isoforms.

3.2.8 Validation of two new I3 antibodies.

The somatic and testicular isoforms of I3 differ at their N-termini. We had previously generated antibodies against two different epitopes of I3 – I3 peptide 1 and peptide 2 (Figure 47). I3 peptide 1 antibody should recognize only the somatic isoform I3 peptide 2 antibody should recognize both isoforms. An I3 variant antibody was raised against the third epitope in red and should recognize both isoforms. NT I3 antibody was raised against the epitope shown in blue. This antibody should recognize only the testicular isoform (Figure 47).

Figure 47: Protein sequence of somatic isoform and predicted sequence of testicular isoform of PPP1R11 (I3) indicating the epitopes against which the I3 antibodies were raised.

A B

Figure 48: Validation of N-terminus (NT) I3 antibody. This antibody was raised against the N-terminus of the predicted amino acid sequence of testicular isoform of I3 and should recognize only the testicular isoform and not the somatic isoform of I3. The antibody recognizes a unique band at approx. 23 kDa, which could be the testicular isoform of I3, in both testis (T) and heat-stable testis extracts (T*) but not in heat-stable brain extracts (B*). Antibody was used at 1:1000 dilution.

Figure 49: Validation of I3 variant antibody. This antibody should recognize both isoforms of I3 but it shows only the somatic isoform of I3 at 27 kDa. Antibody was used at 1:1000 dilution.

The NT I3 antibody recognizes a band at approx. 20kDa in testis protein lysates

(T), a band at approx. 24kDa in His-tagged recombinant testicular I3 and is non-reactive against the recombinant ubiquitously expressed isoform of I3. The 20kDa band is absent in heat-stable testis protein extracts (T*) suggesting that it is a non-specific band. The

23kDa band was observed in T and T* but not in heat-stable brain protein extracts suggesting that this could be the testis I3 protein (Figure 48). The I3 variant antibody should recognize both isoforms of I3 since it was raised against an epitope present in both isoforms. The antibody recognizes the His-tagged recombinant isoforms of both isoforms but recognizes only the endogenous ubiquitously expressed isoform of I3 in Hb+ testis protein lysate (T) and heat-stable testis protein extracts (T*) (Figure 49). The reason why

this antibody does not recognize the putative 23 kDa testis form seen with the NT I3 antibody is not known. Using these new antibodies we proceeded to purify testis and sperm extract and identify by microsequencing the testis isoform of I3.

3.2.9 Identification of testicular isoform of I3 in testis and sperm.

We first tried to purify and identify the testicular isoform of I3 from testis and sperm. Testis and sperm protein extracts were purified using DEAE and analyzed using

I3 peptide 2 antibody. The fractions containing I3 were separated on SDS-PAGE and microsequenced following trypsin digestion (Figure 39 bands 2 and 3). The sequencing did not, possibly due to low abundance, show the presence of either isoforms of I3. We next performed the purification using heat-stable protein extracts. During the validation of the new NT I3 antibody we realized that, for whatever reason, testis I3 was present in lower amounts in extracts when compared to the ubiquitously expressed I3. Hence, we had to perform our purification using highly concentrated testis protein lysates. The

DEAE purified fractions were analyzed by Western blot using I3 peptide 2 antibody

(should recognize both isoforms) and NT I3 (recognizes only the testis isoform).

Figure 50: DEAE purification of heat stable testis extracts. A 23kDa band corresponding to the testicular isoform of I3 was identified in the “Peak 1” fraction.

Western blot analysis of DEAE purified heat stable testis extracts with both I3 peptide 2 and NT I3 revealed the presence of a 23kDa band in “Peak 1” fraction. This 23 kDa is presumably the testis isoform. This fraction will be used for microsequencing.

Since, the testis I3 is present in lower amounts we wanted to further enrich it by immunoprecipitating the “Peak 1” fraction using the isoform specific NT I3 antibody.

Figure 51: NT I3 immunopreciptation of “Peak 1” fraction from DEAE Sepharose purification.

Immunoprecipitation of “Peak 1” fraction by NT I3 antibody shows that the

23kDa band has been pulled down and the corresponding IgG control showing no band at

that position. The immunoprecipiation fraction was separated by SDS-PAGE, the band at the appropriate position will be excised for microsequencing.

3.2.10 Presence of a unique transcript of sds22.

A previous report had identified a unique transcript of sds22 that was specifically and highly expressed in rat testis (65). Examination of mouse EST database and recent gene annotation of Ppp1r7 gene in mice suggests that a similar transcript could be produced in mice due to alternate splicing.

Figure 52: Schematic representing the exon-intron arrangement and alternate splicing of Ppp1r7. The exons and introns are not drawn to scale.

Figure 53: Comparison of amino acid sequence between regular and unique isoform of sds22. The two isoforms differ in the C-terminus. The testicular isoform has a longer C- terminus due to alternate splicing.

The regular transcript of sds22 contains ten exons and nine introns and is approximately 3.5kb in length. The unique sds22 transcript contains eleven exons and ten introns that would result in a unique C-terminus tail in the predicted amino acid sequence.

3.2.11 The unique 0.9kb message of sds22 is present only in testis.

Figure 54: Tissue northern blot of total RNA (25µg) demonstrating the specific expression of a unique message for sds22 in testis when probed with 3’ sds22 probe.

The predicted length of the transcript for the unique isoform of sds22 is approximately 1.1kb. The 1.1kb band observed in the tissue Northern blot is seen to be present largely in testis. Henceforth, this isoform will be referred to as the testicular isoform of sds22.

Figure 55: Agarose gel electrophoresis of alternate isoform of sds22 PCR product containing a unique 3’UTR.

Figure 56: Confirmation that the testicular isoforms of sds22 is present in testis by RT- PCR using specific primers (arrow-heads).

A band at approximately 1.1kb was observed when the PCR product was analyzed by gel electrophoresis. This 1.1kb band included exon 11 and the unique

3’UTR. The PCR product was purified, sub-cloned and sequenced. Sequencing of the

1.1kb fragment confirmed that the testis isoform of sds22 contained an additional exon

(exon 11) a unique 3’ UTR due to alternate splicing (Figures 52 and 56).

Figure 57: Predicted amino acid sequence of testicular isoform of PPP1R7 (sds22) indicating the epitopes against which antibodies were raised. Both antibodies should recognize both isoforms of sds22.

The sds22 antibodies that we had generated should recognize both isoforms. An isoform-specific antibody can be generated based on the predicted amino acid sequence.

But we have not yet undertaken this task. As described below, efforts are underway to purify by FPLC and identify the protein by MS.

3.2.5 Identification of testicular isoform of sds22 in testis and sperm.

To identify the testiscular isoform of sds22 in testis and sperm, we purified mouse testis and sperm protein lysates using FPLC and analyzed the different fractions by Western blot. The antibody used for Western blot analysis of the purified fractions should recognize both isoforms of sds22.

Figure 58: Identification of testicular isoform of sds22 in mouse testis. A. DEAE purification of mouse testis protein lysate. B. Mono Q purification of “Peak 1” and “Peak 2” fractions from DEAE purification.

Figure 59: Identification of testicular isoform of sds 22 in mouse sperm. Mouse sperm protein lysate was purified using DEAE Sepharose.

“Peak 2” fraction from Mono Q purification of mouse testis (Figure 59) and

“Peak 2” fraction from DEAE Sepharose purification of mouse sperm (Figure 60) were separated on SDS-PAGE and the bands at the appropriate positions were chosen for analysis by microsequencing.

3.2.5 Developmental expression of unique testicular isoforms.

To determine the stage of spermatogenesis at which the message for the testis- specific isoforms were expressed, total RNA and protein extracts were prepared from mouse testis at various days post partum (dpp). Testis at 3-6 dpp mainly contain Sertoli cell and undifferentiated spermatogonial stem cells. During 7-10 dpp spermatogonial stem cells undergo mitosis in testis and during 10-14 dpp type B spermatogonia differentiate to form primary spermatocytes and meiosis I commences. At 15-19 dpp

meiosis II begins and secondary spermatocytes are formed. Haploid post-meiotic round spermatids are formed at 18-22 dpp. Mature spermatozoa are present in the testis around

25-30 dpp. Collecting testis from mice at these various time points represent the various developmental stages of spermatogenesis and is useful in ascertaining the stage of spermatogenesis at which this unique message is expressed. Northern blot analyses reveal that the unique message for the testicular isoforms is expressed post-meiotically (Figures

60-62). Western blot analyses of protein extracts of mouse testis from similar time points also reveals that these proteins are expressed post-meiotically (Figures 63-65).

Figure 60: Northern blot analysis of total testis RNA (25µg) demonstrating the developmental expresstion of I2 isoforms when probed with cDNA of somatic isoform of I2. The testicular isoform of I2 is highly expressed postmeiotically.

Figure 61: Northern blot analysis of total testis RNA (25µg) demonstrating the developmental expression of I3 isoforms when probed with cDNA of somatic isoform of I3. The testicular isoform of I3 is highly expressed postmeiotically. The bands observed above and below the expected position of somatic I3 were due to reprobing the blot with other probes.

Figure 62: Northern blot analysis of total testis RNA (25µg) demonstrating the developmental expresstion of sds22 isoforms when probed with cDNA of somatic isoform of sds22. The testicular isoform of sds22 is highly expressed postmeiotically.

Figure 63: Developmental expression of I2. Western blot analysis of testis protein lysate (20µg) demonstrating the post-meiotic expression of I2.

Figure 64: Developmental expression of I3. Western blot analysis of testis protein lysate (20µg) demonstrating the post-meiotic expression of I3.

Figure 65: Developmental expression of sds22. Western blot analysis of testis protein lysate (20µg) demonstrating the post-meiotic expression of sds22.

Northern and Western blot analyses reveal that the message and protein of

I2, I3 and sds22 are produced post-meiotically which matched the expression pattern of PP1γ2. This co-ordinated expression of PP1γ2 and its testis-specific regulators is a very interesting finding. To understand the possible role that PP1γ2 could be playing in regulating the expression of its binding partners we decided to examine their levels in testis of Ppp1Cc -/- mice.

3.2.9 Expression levels of PP1γ2 binding proteins I2, I3 and sds22 in Ppp1Cc -/- mice.

To understand the role of PP1γ2 in regulating the levels of I2, I3 and sds22,

Northern blot analyses of total RNA from Ppp1cc -/- testis and wild-type mice was performed to compare the levels of their transcripts. To detect the testis I2 message a full- length testis I2 was used as probe. This probe will identify messages for both the

isoforms of I2. To compare the message for testis I3, an isoform-specific probe corresponding to the 5’ UTR of the testis I3 was used. For sds22, the probe used was against the region from a portion of exon 10 to the unique 3’UTR. This probe should recognize both isoforms of sds22. The protein levels of I2, I3 and sds22 were also compared in Ppp1Cc -/- and wild-type testis to check if there was a co-relation between the data from Northern blot.

Figure 66: Northern blot analyses of total RNA (25µg) from testis of wildtype compared with Ppp1Cc -/- testis demonstrating reduced expression of testicular isoform message of I2 (A), I3 (B) and sds22 (C). The lower panel represents the corresponding actin (loading control) blots.

Figure 67: Western blot analyses of testis protein lysate (20µg) of wildtype compared with Ppp1Cc -/- testis demonstrating reduced expression of I2, I3 and sds22.

Northern blot comparing the message levels of I2 revealed that the transcript for testis I2 was decreased in Ppp1Cc -/- testis when compared to wild-type whereas, the transcript level for the ubiquitously expressed I2 was unaltered. The same was observed in the case of I3 and sds22 message, their transcript levels were decreased in Pp1Cc -/- mouse testis (Figure 66). The message for the ubiquitously expressed isoform of sds22 was not detected. Comparison of I2, I3 and sds22 protein also revealed the same pattern, protein levels were decreased in Ppp1Cc -/- mouse testis (Figure 67). This finding suggests that PP1γ2 could be playing a role in regulating the levels of I2, I3 and sds22 at the transcription level by regulating a transcription factor(s) that play a role in I2, I3 and sds22 transcription.

Summary

We have identified testis specific transcripts for three ubiquitously expressed PP1 binding proteins – I2, I3 and sds22. We also show that these transcripts are expressed post-meiotically during spermatogenesis that is similar to Pp1γ2 expression. For the first time we have conclusively shown the presence of both the somatic and testicular isoforms of I2 protein in testis and sperm. Efforts to identify the testicular isoforms of I3 and sds22 by protein sequencing are underway. Pp1Cc -/- compared to wild type testes contain decreased levels of I2, I3 and sds22 message and protein. These findings suggest that PP1γ2 itself could be involved in regulating the expression of its binding partners.

3.3

Aim 3: To initiate studies in order to identify possible PP1γ2 substrates.

Rationale:

Male mice lacking Ppp1Cc gene are infertile due to the lack of mature sperm.

Rescue of spermatogenesis but not infertility occurs in Ppp1Cc null mice transgenically expressing low levels of PP1γ2 (81). Infertility is due to malformed sperm as seen in

Figure 68.

Figure 68: DIC images of A. testicular B. caput and C. caudal spermatozoa displaying structural defects in head, mitochondrial sheath and at the midpiece/principal piece junction. (100X magnification) (81).

A goal of this aim is to determine the basis for the malformed sperm. The possibility is that defective sperm lack selected proteins or that the levels of selected proteins are altered in testis leading to defective sperm morphogenesis. Another possibility is that signaling mechanisms operating during the final steps of spermatogenesis could be compromised. Complete absence or low levels of the serine/threonine phosphatase in sperm should result in the hyper-phosphorylation of proteins. Analysis of phosphoproteome of malformed sperm could aid in the identification of possible PP1γ2 substrates. Before undertaking the phosphoproteome analysis, I wanted to first compare the proteome of defective sperm from transgenic mice with normal sperm from Ppp1Cc +/- mice. I used 2D-DIGE for this analysis. 2D-DIGE

using fluorescent dyes is a technique that enables analysis of up to three samples in the same gel. The samples are labeled with fluorescent dyes prior to electrophoresis. The dyes – Cy2, Cy3 and Cy5, are spectrally distinct and are designed to be of exact size and charge. Since these dyes are of the exact same size and charge the position of proteins tagged with the dyes on the gel should remain unaltered. The ability to run multiple samples on the same gel reduces variations that arise from individual gel runs.

3.3.1 2D-DIGE analysis comparing the relative expression of sperm proteins from

Ppp1Cc +/- and Ppp1Cc -/- expressing Ppp1Cc2 under the Pgk2 promoter (rescue mice).

Global protein abundance from wild type and PP1γ2-rescue sperm was compared by Difference Gel Electrophoresis (DIGE). Protein extracts from wild type and rescue sperm were labeled with the fluorescent dyes Cy3 and Cy5. The two dye-labeled samples were mixed and run on a 2-D gel followed by analysis of the protein spots using the

Decyder software (GE/Amersham). In the first run we compared sperm from Pgk2-driven

PP1γ2 rescue line (where males are infertile) A-line (highest expresser of the Ppp1Cc2 transgene) to sperm from a littermate control (Ppp1cc +/-) (Figures 69 and 70). The experiment was repeated with sperm proteins extracted from another line of Pgk2-driven

PP1γ2 rescue line, E-line (the next highest expressing transgenic line where the sperm are malformed and the males are also infertile). The 2D-DIGE results show a similar pattern

(Figures 71 and 72) as the first run. The values indicating the fold difference in protein expression and their approximate molecular weight from the first run are listed in Table

6. A third run was performed with 50µg protein and the spots that had over a 3-fold difference were analyzed by tandem mass spectroscopy following proteolysis. An intriguing finding was that a majority of the proteins that had altered expression in malformed sperm were enzymes involved in metabolic pathways such as glycolysis

(Figure 73) and gluconeogenesis (Figure 74). This was unexpected since, we had expected changes in sperm structural proteins to be altered in malformed sperm (Figure

69). The complete list of proteins identified is shown in Appendix IV.

Figure 69: 2D-DIGE analysis of Ppp1Cc +/- (green) and Ppp1Cc -/- transgene positive (A-line) (red) mouse sperm.

Figure 70: Differential gel electrophoresis (DIGE) of sperm extracts from Ppp1cc +/- (labeled with Cy3) and sperm extracts from Pgk2-driven rescue mice (labeled with Cy5). The red and green spots represent proteins up- and down-regulated in rescue when compared to Ppp1 cc +/- and the yellow spots represent proteins that are equally abundant.

Figure 71: 2D-DIGE analysis of Ppp1Cc +/- (green) and Ppp1Cc -/- transgene positive (E-line) (red) mouse sperm.

Figure 72: 2D-DIGE overlay showing the differential expression pattern of proteins between Ppp1Cc +/- and Ppp1Cc -/- with transgene positive (next highest expresser) with the spots that were measured demarcated. The two 2D-DIGE runs show a similar pattern.

Table 6 : The spots that were analyzed for change in expression levels with approximate molecular weights of spots that had over a 3-fold change in expression.

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Figure 73: Glycolytic enzymes that were either up regulated or down regulated in the transgenic mouse.

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Figure 74: Gluconeogenic enzymes that were altered in in sperm extracts. This is first evidence of the presence of a gluconeogenic enzyme in sperm. The other proteins that were identified are listed in Appendix IV.

Summary

We have initiated studies to identify possible PP1γ2 substrates. We used 2D-

DIGE to compare the sperm proteome of Ppp1Cc +/- and Ppp1Cc -/- that transgenically express Ppp1Cc2 under the control of the Pgk2 promoter (rescue mice). These rescue mice express lower amount of PP1γ2 than wild type mice and sperm from these transgenic mice display gross morphological defects (Figure 69) resulting in lack of motility and infertility. The initial comparison of the sperm proteome identified a host of proteins whose levels were altered in defective sperm. The interesting finding was the

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observation that glycolytic enzymes and gluconeogenic enzymes were altered in malformed sperm. The identification of the glucoenoegenic enzyme fructose 1,6- bisphosphatase suggests that gluconeogenesis may occur in sperm. These preliminary findings have to be further confirmed by analyzing malformed sperm from other transgenic lines. These findings must also be confirmed by other independent approaches such as western blot analysis.

Chapter 4

Discussion

Role of RSPH1 - Aim1

RSPH1 was thought to be present in two isoforms – P44 that is expressed in the testis and that P30 is expressed in the epididymis (78). The epididymal isoform was apparently formed as a result of exon 2 being alternatively spliced. This was determined using RT-PCR and isoform specific antibodies (78). When protein extracts made from different regions of the epididymis (caput, corpus and cauda) was analyzed by SDS-

PAGE and western blotting using the C-terminus antibody, P30 was expressed only in the corpus and caudal epididymydes and not in the corpus epididymis (Figure 13). This finding suggested that the epididymal isoform could be playing an important role in epididymal maturation of sperm. These results implied that P30 secreted by the epithelium in the corpus and caudal regions of epididymis is incorporated into sperm during their passage through the epididymis. However the possibility that P30 arose from proteolysis of P40 present in sperm could not be ruled out. To examine these possibilities epididymis from mice lacking sperm were analyzed. Epididymis of Ppp1Cc knockout mice is devoid of sperm and was an ideal model to employ for this analysis. When protein extracts from caput, corpus and caudal regions of the epididymis of the null mice were analyzed using SDS-PAGE and western blotting using the C-terminus antibody, the results were identical to that of the wild type mice epididymis, that is P30 was expressed

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in the corpus and caudal regions and not in the corpus region of the epididymis (Figure

13). This further strengthened the hypothesis that the P30 isoform of RSPH1 was being secreted by the epididymal epithelium into the luminal fluid and was taken up by the sperm during epididymal transit.

The amount of luminal fluid obtained from mouse epididymis is limited.

Furthermore, it is difficult to obtain luminal fluid free of contamination with sperm. To overcome this problem and to further analyze the role of P30, we decided to use bovine epididymis where substantial amounts of epididymal fluid relatively free form contamination with sperm can be obtained. A new antibody had to be made which would detect bovine RSPH1 since the RSPH1 antibodies that were available could not recognize the bovine homolog (Figure 14).

The differential expression of P30 in the epididymis suggests a possible role for this isoform in epididymal maturation of sperm. That is, P30 acquired by sperm from the epididymis could be responsible for the acquisition of motility and fertility by sperm during their passage through the epididymis. To answer this question I decided to transgenically express the testicular isoform of RSPH1, P44 on the Rsph1 knockout background. These transgenic mice will express P44 in testis but not P30 in the epididymis – the equivalent of generating an epididymis-specific RSPH1 knockout mice.

(Figure 9).

During the validation of the new bovine RSPH1 antibody, which was designed to identify both isoforms in bull and mouse, we observed that this antibody detected only

P44 but not P30 even though it was supposed to recognize both isoforms. Moreover,

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examination of the epididymis of Rsph1 knockout mice which are expected to lack all protein products derived from the gene, surprisingly we found that P30 was present in the mutant mice (Figure 16). This raised the unexpected possibility that P30 was not related to RSPH1. To confirm this, P30 was purified from epididymis using FPLC and the purified protein was micro-sequenced (Figures 17-19) by tandem-MS.

Microsequencing analysis showed that P30 was not related to RSPH1 but was in fact another epididymal protein – CRISP1 (Figures 20 and 21).

Cysteine rich secretory proteins (CRISP) belong to a superfamily of proteins known as CAP (CRISP, antigen 5 and Pr-1). These proteins are mainly found in the male reproductive tract of vertebrates and in the venom of poisonous reptiles (82). There are four different kinds of CRISPs expressed in mice and they play a role in almost every stage of sperm maturations. CRISP1 and CRISP4 are expressed in the epididymis by the principal cells and get incorporated into sperm during epididymal transit. They are thought to play a role in keeping the sperm in a “dormant” state in the epididymis and are thought to play a role in decapacitation (82). The amount of CRISP gets diluted in the female reproductive tract and their diffusion off sperm helps in the initiation of certain signaling pathways required for capacitation (83). CRISP1 gets irreversibly associated with the sperm plasma membrane around the head during epididymal transit (82-85) and is present in the sperm head after the acrosome reaction has taken place (86). Crisp1 knockout male mice produce normal sperm and are fertile and produce normal size

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CRISPs in sperm function AJ Koppers et al 113

Figure 1 Diagrammatic representation of CRISP expression and function in male fertility. CRISP2 is expressed in haploid germ cells, wherein it is incorporated into the growing acrosome and sperm tail. Within the testis, it has been proposed that CRISP2 is involved in germ cell–Sertoli cell adhesion, and in the tail, it has been proposed to be involved in regulating flagellar beating via its ability to regulate ryanodine receptors. CRISP2 remains associated with the fusogenic region of the sperm head after the acrosome reaction. CRISP1 and 4 are both expressed by the principal cells of the epididymis and become incorporated into the maturing spermatozoa and have been implicated as a decapacitation factor. CRISP3 is excreted from the prostate and seminal vesicles and forms part of the seminal plasma. Although variations in CRISP3Figure sperm content 75 and: Schematic sequence have beenrepresenting correlated with fertility,the localization no defined role is currently and function known. CRISP, of cysteine-rich the different secretory protein. kinds of pachyteneCRISPs spermatocytes; in male however, reproductionvia the activity (Ref of the 82). RNA-bind- relation to sperm motility later in this review. Unlike the other ing protein DAZL, Crisp2 mRNA undergoes a period of translational CRISP genes, Crisp2 is not induced by androgens.39 delay before the initiation of translation in round spermatids.33–37 As The first hint at CRISP2 function came from a study by Maeda et shown by the staining (Figure 2), mouse CRISP2 was localized in al.40 who used a Jurkat cell transfection assay to identify CRISP2 as round spermatids through to elongated spermatids, wherein the pro- having the potential to promote fusion between Jurkat and Sertoli tein becomes included into the developing acrosome and the connect- cells. Further, they showed that CRISP2 antisera were able to interfere ing piecelitters and outer but dense their fibers sperm of the spermhave tail. been34–36,38 shownThe potential to havein thea reduced cell adhesion ability between to germ penetrate and Sertoli the cells. zona Using deletion function of CRISP2 in the connecting piece is further discussed in studies, the adhesive sequence was narrowed to the N-terminal most pellucida and to bind to the oocyte plasma membrane (87). These knockout mice did

display subfertility due to reduced levels of tyrosine phosphorylation after in vitro

capacitation (87). Also, CRISP1 antisera has been shown to interfere with sperm-egg

interaction (88). This suggests that CRISP1 plays an important role in sperm-egg

interaction and could also play a role in capacitation of sperm.

The unexpected finding that P30 was not an RSPH1 isoform ended our quest to

examine the apparent role for P30 in sperm function. However our interest in the role of

RSPH1 in spermatogenesis still remains. Rsph1 and Ppp1Cc knockout male mice are

Figure 2infertileLocalization and of murine display CRISP proteins similar in male phentotype reproductive tissues. (80,44). (a–c) CRISP1 The staining targeted of the caput, deletion corpus and cauda of epididymis,these genes respectively. Staining present in the cytoplasm and stereocilia of the principal cells and spermatozoa within the epididymal lumen. (d) CRISP2 staining of the testis shows protein present within the round and elongated spermatids. (e–g) CRISP4 staining of the caput, corpus and cauda epididymis, respectively. Staining present within the cytoplasm of the principal cells, within the stereocilia and in the epididymal lumen, including within epididymosomes. (h) CRISP3 staining of the prostate gland within the apical aspect of the epithelium and in luminal secretory products. Scale bars5100 mm. CRISP, cysteine-rich secretory protein.

Asian Journal of Andrology 107

results in defective spermiogenesis. During purification RSPH1 co-elutes with PP1γ2 and its binding partners I3, sds22 and 14-3-3 (Figure 22). Native PAGE of purified testis protein fractions shows that RSPH1 co-migrates with PP1γ2, I3 and sds22 (Figure 24) suggesting that RSPH1 could be a binding partner of PP1γ2 and/or a binding partner of

PP1γ2 regulating proteins. It has also been shown that RSPH1 interacts with sperm 14-3-

3, which is a known PP1γ2 binding partner (71,72). These results suggest that RSPH1 could play a possible role in regulating PP1γ2 function either by interacting directly with

PP1γ2 or by forming a complex with one or more of the PP1γ2 binding partners.

Due to this possible interaction between Rsph1 and PP1γ2, testis from Rsph1 knockout mice were analyzed to see if levels of PP1γ2 and its binding partners were altered. Western blot analyses showed that levels of PP1γ2 I3, sds22 and 14-3-3 were not different in Rsph1 knockout mice. However the reduced levels of these proteins in protein extracts from the epididymis of Rsph1 knockout mice is due to the absence of sperm in the epididymis (Rsph1 KO) (Figure 23). Although RSPH1 binds to the PP1γ2 binding partners, whether RSPH1 actually plays a role in regulating PP1γ2 activity is still unclear.

RSPH1 is localized to the radial spokes of the axoneme in the sperm flagella (79).

Radial spokes are important components of the axoneme and play a vital role in regulating the dyenin arms that produce motility by bringing about the sliding of microtubules (24). The importance of radial spokes has been studied in Chlamydomonas, where the lack of radial spokes results in paralyzed flagella (89). During the proteomic analysis of radial spokes in C.intestinalis, a unique 116kDa protein containing a

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calmodulin binding IQ motif and a ubiquitin domain – CMUB116 was identified as a possible binding partner of radial spoke protein MORN40 (89), an ortholog of RSPH1.

Examination of expressed sequence tags (EST) database showed that the mammalian ortholog of CMUB116 is highly expressed in testis (Figure 28). Immunoprecitpiation of mouse testis extracts demonstrated the interaction of RSPH1 and CMUB116 (Figure 29).

Considering the localization of both proteins to the radial spoke of the axoneme, the homolog of CMUB116 could be playing an important role during flagellar assembly in spermiogenesis and in motility of sperm. Our study lays the basis for further studies on the relationship between CMUB116 and RSPH1.

The isoleucine-glutamine rich IQ motif is involved in binding to calmodulin

(CaM). CaM is an intracellular sensor of calcium levels and plays important roles in many important cellular processes. Calcium plays an important role in spermatogenesis

(29,91), spermiogenesis (92) and fertilization (93). Proteins that bind to calmodulin have three different kinds of binding motif; these motifs comprise of two Ca2+- dependent binding of CaM binding proteins and the IQ motif that is Ca2+ independent (92,94).

Certain proteins containing the IQ motif can bind to CaM is both a Ca2+ dependent and independent manner (94). CaM is expressed ubiquitously and its downstream targets determine its cell-specific role (92). In testis there are multiple calmodulin binding proteins. These include calspermin, testis specific calcineurin B, CaMKIV (91), NYD-

SP5 (93) and mtIQ1 (94). These proteins have testis specific expression and are expressed in a stage specific manner. The presence of an IQ motif in CMUB116 suggests that it could play an important role in spermatogenesis.

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Apoptosis prevents overproduction of germ cells to ensure that the nurturing capacity of Sertoli cells is not overwhelmed. Germ cell death by apoptosis occurs under normal conditions (95). It is an important regulatory process that occurs during spermatogenesis which helps in regulating the numbers of different cell types (95). An important mode of apoptosis is ubuiquitination of protein that tags them for degradation by 26S proteosome. Ubiquitination of proteins is determined by the interplay of enzymes that ubiquitinate and deubiquitinate them. Ubiquitin-dependent protein degradation plays an important role in spermatogenesis (95). The rate of ubiquitination is high is testis containing haploid spermatids (96) and the amount of ubiquitin-protein conjugates are highest in elongating spermatids (97,98) and the activity of 16S proteosome is highest at the same stage (99). 26S proteosome plays an important role during spermiogenesis in the formation of the periaxoneme of the sperm tail (99). The presence of a ubiquitin domain in CMUB116 suggests a possible role in sperm morphogenesis during spermiogenesis.

Testis specific protein isoforms – Aim2

Spermatogenesis is a complex and tightly regulated process. One of the means by which spermatogenesis is thought to be controlled is by a specific mode of gene expression. During spermatogenesis somatic forms of proteins are replaced by germ-cell specific isoforms. The transcripts for these germ-cell specific proteins are produced either by alternative splicing or are transcribed from independent duplicated genes. This switch in gene expression from a somatic pattern to a germ cell specific pattern is thought to be vital for spermatogenesis.

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An example of a testis specific protein isoform is PP1γ2. PP1γ2 is a serine/threonine phosphatase that is predominant in the testis and is the only isoform of

PP1 present in mature sperm (42). Testis contains both isoforms of the alternatively spliced Ppp1Cc gene – PP1γ1 and PP1γ2. PP1γ1 is ubiquitous in other tissues and is expressed only in the Sertoli cells in the testis (unpublished data). PP1γ2 is expressed in developing germ cells and its expression dramatically increases in post-meiotic developing germ cells. Like PP1 in general, PP1γ2 function is regulated by its binding partners. In testis and sperm these binding partners include I2, I3 and sds22 (42).

The presence of alternatively spliced transcripts for I2, I3 and sds22 has previously observed (54,55,65,67). However the exact nature of how they are derived and whether protein products derived from these transcripts were actually independent protein isoforms were unknown. Here we show, for the first time, that the unique transcripts for these three proteins are highly expressed only testis and that these transcripts first appear post-meiotically during spermatogenesis (Figures 61-66). The presence of these alternatively expressed transcripts just before the beginning of spermiogenesis, the final stage of sperm production, is intriguing. This mechanism underlying the switch in expression from the somatic isoforms to a germ cell specific isoform should be interesting to determine. The increase in the levels of these germ cell specific transcripts matches the expression pattern of PP1γ2 (unpublished data) suggesting a role for these isoforms in regulating PP1γ2 action during sperm morphogenesis that takes place during spermiogenesis.

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The alternative transcript of I2 was identified from cDNA spermatid library and testis cDNA (Figures 38 and 39). The testis specific isoform is produced by retention of an intron that is spliced out in the ubiquitous somatic form of the message (Figure 34).

Sequence analysis of the alternatively transcribed message of I2 also reveals alternate polyadenylation sites. This could be a mechanism by which the message is stabilized during spermatogenesis. A similar mechanism is known to occur during the expression of cation-dependent mannose-6-phosphate receptor (11).

Although the presence of I2 in sperm had been substantiated by biochemical assays (46,47); the presence of this protein in testis and sperm was not confirmed. This was due to multiple issues faced with the detection of I2. I2 is present in the insoluble fraction of sperm sonicates which makes extraction of this protein difficult. Also, I2 contains PEST sequences that make it prone to proteolysis (101,102). Detection of I2 was also problematic due to the lack of a suitable antibody. For the first time, we purified I2 from testis and sperm and confirm the presence of both isoforms of I2 using mass spectrometry (Figures 40 and 41).

We generated a new antibody specific to the C-terminus of I2. This antibody should detect only the somatic isoform of I2 (Figures 32 and 33). An isoform specific antibody capable of reacting against the testis I2 isoform is not possible because the testis isoform has only three amino acids that differentiates it from the somatic isoform. Using this new C-terminus antibody we show that the I2 protein expression increases post- meiotically (Figure 64). The pattern of protein expression matches the expression of the

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transcript (Figure 61). However we were not able to determine whether this protein expression is exclusively due to the appearance of the testis isoform.

Another interesting observation we have made is there was reduced amounts of testis specific I2 transcript and I2 protein levels in Ppp1Cc knockout compared to wild type mice. The lack of PP1γ2 in testis appears to reduce the amount of testis I2 (Figures

67 and 68).

With respect to I3, examination of EST database reveals that an alternate isoform of I3 can be produced due to the use of an alternate start site in intron 1 (Figures 42 and

43). This would give rise to a protein that has a unique N-terminus (Figure 44). A similar mechanism where alternate transcription start sites are utilized to give rise to testis specific protein isoforms is observed with a number of proteins including the testis and sperm specific protein kinase A catalytic subunit PKA Cα2 (16). The alternate I3 isoform was isolated from testis cDNA (Figure 46). Sequence analysis revealed that the 5’ UTR is due to the predicted alternate start of the transcript compared to the somatic form of the message (Figure 47) but the 5’ UTR is considerably longer than shown in the Ensembl genome database for I3.

Northern blot analyses revealed that this testis I3 transcript is first expressed in post-meiotic developing germ cells (Figure 62). This pattern of expression is essentially identical to the expression of PP1γ2 and the testis specific I2. Western blot analyses also showed that similar to the transcript there is also a matching increase in the protein levels of I3 in developing germ cells (Figure 65). The new NT I3 antibody that should specifically recognize the testicular isoform of I3 is now available (Figures 48 and 49).

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We need to confirm that the protein that it recognizes is the testicular isoform of I3. Like

I2, the levels of I3 transcript and protein are reduced in Ppp1Cc knockout mice compared to wild type mice (Figures 67 and 68).

Attempts to purify and identify this protein from testis and sperm have so far been unsuccessful. It may be noted that I3 also has PEST sequences that make it susceptible to proteolysis (100,101). However sequencing of a recent purification attempt is in progress.

It is fascinating that the two evolutionarily ancient and ubiquitous heat stable inhibitors of PP1 are present as testis-specific isoforms and display a spatio-temporal expression pattern similar to the testis and mammal specific PP1 isoform - PP1γ2. The same is also true in the case of yet another PP1γ2 binding and regulatory protein - sds22.

Alternate transcripts of sds22 have been previously reported before including the presence of a testis specific isoform in rats (65,67). In silico analyses we have conducted suggests the presence of a similar alternate transcript in mice. Our Northern blot analyses confirm the presence of this alternatively spliced isoform in testis (Figure 55). This testis specific isoform arises due to alternate splicing (Figure 53). The testis-specific isoform of sds22 has a unique C-terminus due to alternative splicing (Figure 54). RT-PCR of testis cDNA further confirmed the identity this alternatively transcribed message (Figures 56 and 57) the expression of which rises in postnatal developing testis (Figure 63). Western blot analysis shows that the protein expression sds22 also matches the expression of the transcript. However the antibody used was not isoform specific (Figure 66), so we are not certain whether the developmental increase in testis sds22 is entirely due to the appearance of the testis specific isoform.

11 4

As with I2 and I3, comparison of Ppp1Cc knockout and wild type mice reveals a reduced expression of sds22 in the former (Figures 67 and 68). Purification and identification of the testis specific isoform has so far been unsuccessful which could again be due to low abundance of this isoform. We are currently awaiting the results of microsequencing from another purification experiment (Figures 59 and 60). Production of an isoform specific antibody would greatly help in the efforts to characterize this testis specific isoform.

The three testis specific binding partners of PP1γ2 vary from their somatic isoforms by only in a few amino acid residues in the C-terminus (I2 and sds22) or in the

N-terminus (I3). The regions required for binding and interaction with PP1 are not altered in the testis specific isoforms. Even though we do not have experimental evidence it is likely that all the three proteins bind to PP1γ2 in a manner similar to the somatic forms. If this is true the testis forms do not differ in their biochemical properties from their somatic counter parts. The catalytic subunit of PKA - PKACα is also expressed as two isoforms.

PKACα2 is sperm specific whereas, PKAα1 is ubiquitously expressed (PKA review). In a recent study the biochemical properties of these two isoforms were compared and they are kinetically similar (Ref). What is the basis for the requirement of the specific isoforms in testis? Moreover, why does this requirement arise only at the later stages of spermatogenesis? It is possible that presence of unique N- or C-terminus in these other testis specific protein isoforms enable them to be targeted to specific regions of sperm.

It is notable that these proteins and their transcripts mimic the expression pattern of their binding partner – PP1γ2 and the reduction in the expression of the proteins and

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transcripts in PP1γ2 null mice . This suggests that PP1γ2 could be playing a role in regulating the levels of these binding partners during spermatogenesis and sperm function.

The somatic isoforms of I2, I3 and sds22 are ubiquitously expressed

(Figure 30) and have important roles in diverse physiological processes. Deletion of these genes should result in embryonic lethality making it difficult to use knockout strategy to study their role in spermatogenesis. We plan to create conditional knockout for I2 and sds22 in testis. This will result in mice lacking both isoforms of the protein during spermatogenesis. The conditional knock out will be created using mice expressing Cre recombinase under the control of protamine promoter. Protamine is expressed post- meiotically, similar to the testis specific isoforms of I2 and sds22. This, approach will provide insights into the roles for I2 and sds22 in spermatogenesis and sperm function.

We also plan to transgenically overexpress I2 and a mutated form of I2 under the control of the protamine promoter. Phenotype analysis of the mice overexpressing these transgenic I2 proteins is in progress.

Protein substrates of PP1γ2 – Aim 3

Protein phosphorylation is result of synergistic action of protein kinases and protein phosphatases. The action of these two groups of enzymes determines the phosphoproteome of a cell. In testis, the serine/threonine phosphatase PP1γ2 is the predominantly expressed isoform of PP1 and the only isoform expressed by mature sperm. Targeted deletion Ppp1Cc in mice results in mice lacking alternatively splice

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isoforms – PP1γ1 and PP1γ2 and results in male infertility due to impaired spermiogenesis (44). The lack of a vital phosphatase will result in an imbalance of the phosphoproteome in sperm and result in hyperphosphorylated proteins. These hyperphosphorylated proteins could be possible downstream targets of PP1γ2.

Comparing the phosphoproteome of sperm from mice lacking PP1γ2 with sperm from wild type mice could identify possible PP1γ2 substrates but this is not possible because

Ppp1Cc knockout mice lack mature sperm.

Spermatogenesis can be restored when Ppp1Cc2 is expressed under the control of

PGK2 promoter in Ppp1Cc knockout mice but they have malformed and immotile sperm(81). Sperm from these mice express lower amount of PP1γ2 (81) and this would result in inefficient dephosphorylation of downstream targets. Since these mice produce sperm and have impaired dephosphorylation it allows for phosphoproteome analysis and identification of possible PP1γ2 substrates.

Before comparing the phosphoproteome, initial experiments to analyze any difference in protein expression using two dimensional difference gel electrophoresis

(2D-DIGE) was performed. 2D-DIGE of sperm from Ppp1Cc +/- mice and from mice expressing transgenic Ppp1Cc2 under the Pgk2 promoter was performed and spots that were differentially expressed were sequenced.

The first run included mice that expressed the highest amounts of the Ppp1Cc2 transgene – A line and the second run included mice that had the next highest expression of the Ppp1Cc2 transgene – E-line. Both these lines display the same gross structural abnormalities in sperm morphology. This was done to demonstrate reproducibility

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between runs. The final experiment, from which the spots were excised, was performed with sperm from A-line.

Since the sperm that were analyzed displayed gross morphological abnormalities, we expected that majority of the proteins that would be identified would be structural proteins. Unexpectedly, however, the analyses revealed that there was a change in the expression levels of enzymes involved in metabolic pathways required for

ATP synthesis, mainly glycolysis. The spots that were chosen for microsequencing were differentially expressed - either up- or downregulated, by at least 3-fold in malformed compared to normal sperm. The microsequencing results revealed that the proteins altered were largely metabolic enzymes. The majority of the enzymes are part of the glycolytic pathway (Figure 64). Out of the ten enzymes that are involved in glycolysis, six of them were misregulated in deformed sperm. Among the six enzymes that were identified, three enzymes are involved in the “pay-off” phase of glycolysis, during which

ATP and NADH are generated. These enzymes are GAPDH, phosphoglycerate kinase and pyruvate kinase. Another interesting finding in this aim is the identification of the gluconeogenic enzyme – fructose 1,6-bisphosphatase. This is a key enzyme involved in gluconeogenesis . There have been no previous reports for the operation of gluconeogenesis pathway in spermatozoa. The identification of this enzyme suggests new avenues by which glucose metabolism and ATP synthesis could occur in sperm. The set of other proteins that were identified during this preliminary analysis are listed in

Appendix IV.

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Generation of ATP is vital to sperm function for it is the energy currency responsible for sperm motility. The sperm midpiece contains mitochondria, the site for oxidative phosphorylation and ATP synthesis. The localization of machinery required for oxidative phosphorylation within the midpiece is very interesting. This compartmentalization suggests that ATP synthesized in this region would have to be transported to the flagella by diffusion to be utilized to generate motility (102). It was observed that diffusion was sufficient to transport ATP to the tip of the flagellum in bull and sea urchin sperm (102) although, there was doubt regarding the validity of this model in rodents which have longer sperm (103). Another model that described a linear relationship between flagellar length and mitochondrial volume could explain diffusion as a means of ATP transport in rodents (104). An interesting observation was that when sperm were treated with oxidative phosphorylation uncouplers their motility was unaffected (105). Sperm overcome this by utilizing glycolysis for ATP synthesis and the importance of glycolysis was realized when a sperm specific isoform of glyceraldehyde-

3-phosphate dehydrogenase (GAPD-S) was knocked out. Male mice lacking Gapds were infertile whereas the females were normal and sperm from these mice were immotile and the intracellular ATP concentration was 10% of wild type sperm (107). The importance of oxidative phosphorylation cannot be ruled out based on these observations. There have been suggestions made that ATP generated from oxidative phosphorylation could diffuse to the head (107) whereas, glycolysis is responsible for ATP generation in the tail.

My results have to be confirmed by analyzing other mice lines expressing different amounts of transgenic Ppp1Cc2. This could strengthen the preliminary findings

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shown here. Also, western blot analysis comparing the levels of these proteins is essential

to confirm results from DIGE and protein sequencing. Identification of the

phosphoproteome should also help in identification of PP1γ2 substrates. .

Phosphoproteome analysis of testis from Ppp1Cc mice using 2D-gel

electrophoresis and ProQ Diamond staining identified ten hyperphosphorylated proteins

(109). Of these ten identified proteins HSPA2, which is expressed from pachytene

spermatocytes to elongated spermatids, has been shown to interact with PP1γ2 by

immunoprecipitation and pull-down assays (109).

Another approach to identify downstream targets of PP1γ2, in progress in our

laboratory, is by using a chemical genetics approach. This approach employs using a

mutated form of a kinase that uses an ATP homolog (ATPγS). The mutated kinase uses

the ATP homolog as the phosphate moiety donor to thiophosphorylate its target proteins.

This phosphate moiety is then alkylated by the addition of p-nitrobenzylmesylate

(PNBM) and a specific thiophosphate-ester antibody can detect the phosphorylated target

proteins (109).

Allen et al. Page 10 HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscript

Figure 1. Strategy for labeling individual kinase substrates. (a) Reaction sequence for affinity tagging AS kinase substrates. First, an AS kinase (magenta) uses N6-alkylated ATPS (A*TPS) to thiophosphorylate its substrates (pale magenta). In a second step, alkylation with PNBM yields thiophosphate esters and thioethers. Only AS kinase substrates are recognized by -hapten– IgG. (b) Structure of the hapten conjugate used to elicit thiophosphate ester specific antibodies. (c) JNK1 was incubated with c-Jun–GST and combinations of ATPS and PNBM as indicated; reaction products were analyzed by western blot with -hapten–IgG. -GST–IgG confirms equal loading. Full-length blots are available in Supplementary Figure 1b.

Nat Methods. Author manuscript; available in PMC 2010 September 2. 120

Figure 76: Schematic demonstrating the phosphorylation reaction using a mutated kinase and identification of its substrates using specific antibodies (Ref 109).

This experiment will be performed using sperm and testis from ATP-analog sensitive PKA knock-in mice (110). These mice express a mutated form of PKA Cα

(M120A) and do not express wild type PKA Cα (110). This mutation enlarges the binding site of ATP on the kinase allowing it use ATP homologs as γ-phosphate donors and also making the kinase susceptible to novel inhibitors (109). This would enable us in identifying proteins that are phosphorylated by PKA in testis and sperm.

The PKA holoenzyme comprises of two regulatory (R) and two catalytic (C) subunits. The importance of cAMP and cAMP-mediated signaling for sperm function has been well documented (43). A downstream target of cAMP is PKA. The different PKA subunits RIα, RIIα, Cα1 and Cα2 are differentially expressed during spermatogenesis

(16). RIα is expressed throughout spermatogenesis and RIIα is the predominant subunit expressed in sperm during spermiation (16). Cα1 and Cα2 are alternaltively spliced products differing in their N-terminus. Cα1 has a unique 14 amino acid residue whereas

Cα2 has a unique 7 amino acids because of a different transcription start site (16). The expression pattern of these two isoforms also varies during spermatogenesis. Cα2 starts being expressed during the pachytene stage of spermatogenesis (16). This expression pattern coincides with the expression of PP1γ2 and makes PKA a possible candidate that could phosphorylate PP1γ2 substrates.

121

The dimerized regulatory subunits bind to specific scaffolding proteins known as

A-kinase anchoring proteins (AKAP) that help in localizing PKA and thus enabling phosphorylation of downstream targets. AKAPs are present in multiple isoforms and three of these isoforms are testis specific and are present in the fibrous sheath (FS) of mature sperm. The FS is the longest component of the sperm flagellum and defines the principal piece and is unique feature observed only in mammals and some species of birds (3). The most abundant protein present in the FS is AKAP4. The expression of this protein is another example demonstrating the spatio-temporal expression of proteins during spermatogenesis. The Akap4 gene is transcribed in round spermatids but the message is translated only during the development of elongating spermatids (111-113).

AKAP4 has two PKA-anchoring domains – one that can bind to either RIα or RIIα and the other that only binds to RIα (113). Sperm from Akap4 knockout mice have shortened flagella compared to sperm from wild type and lack progressive motility (114). Another isoform of AKAP that is present in FS and that is testis specific and expressed postmeiotically is AKAP3 (AKAP110) (115). Akap3 is also transcribed during the development of round spermatids and the message is translated during spermiogenesis

(3). AKAP3 has only one PKA-anchoring domain and binds to RIIα. AKAP4 binds to

AKAP3 and AKAP3 binds to ropporin that interacts with members of the Rho-signaling pathway (116). The third isoform of AKAP present in the FS is TAKAP-80. It was identified in rat and was also expressed during spermiogenesis (3) and contains only a single RIIα binding site (3).

122

The presence of these testis-specific isoforms of AKAP and its abundance in FS highlights the importance of PKA-mediated signaling in spermatogenesis and sperm function and consolidates the fact that it possibly could be the kinase that phosphorylates

PP1γ2 substrates.

Proteins that are identified from both approaches – sperm phosphoproteome analyses of mice expressing transgenic Ppp1Cc2 and from ATP analog-sensitive PKA knockin mice are likely substrates for PP1γ2.

In summary, in Aim 1 I made the discovery that P30 considered to be RSPH1 isoform is erroneous. I also discovered the possibility of the interactions of RSPH1 with

PP1γ2 and its regulators and with a new protein CMUB116. These interactions could be basis for the functions of PP1γ2 and RSPH1 during spermiogenesis. Perhaps the most interesting and significant discovery of my research is the finding that all three ubiquitous and essential PP1 regulatory proteins, I2, I3 and sds22, are expressed as testis specific isoforms. Moreover, the spatio-temporal expression of the transcripts and proteins matches the expression of the testis, sperm and mammal specific PP1 isoform PP1γ2.

Finally, in the third aim, my studies have laid the basis for the important future studies to identify the substrates for PP1γ2 and for the identification the sperm phosphoproteome involved in the maintenance of the normal sperm structure and function.

Chapter 5

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APPENDIX I

Proteins Identified By Microsequencing Of cDEAE Purified Testis And Sperm

Protein Extracts

Report 1818 Vijay

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Table 2. Summary of results for data dependent experiment. Investigator Digest Identification: Peptides # Mascot sample sample (coverage) score name number 1 cDEAE Ts SB12-94-5 annexin A5 (6753060, 36 kDa) 14 (47%) 1478 band 1 phosphoglycolate phosphatase (40254507, 35 11 (39%) 1147 kDa) EF hand domain containing 2 (31981086, 27 12 (50%) 1102 kDa) lactate dehydrogenase 3, C chain, sperm 9 (30%) 882 specific (7305229, 36 kDa) splicing factor, arginine/serine-rich 2 (6755478, 3 (16%) 695 25 kDa) proliferating cell nuclear antigen (7242171, 29 6 (30%) 568 kDa) heat shock 70kDa protein 4 like (40254361, 95 7 (10%) 468 kDa) C-terminal fragment Pyrophosphatase (27754065, 33 kDa) 9 (28%) 463 cytokine induced protein 29 kDa (13384730, 24 9 (46%) 415 kDa) triosephosphate isomerase 1 (6678413, 27 kDa) 2 (11%) 399 nascent polypeptide-associated complex alpha 3 (19%) 390 polypeptide (41350312, 23 kDa) Charged multivesicular body protein 4b 7 (28%) 380 (109471140, 29 kDa) carnitine deficiency-associated gene expressed 3 (36%) 336 in ventricle 3 isoform CDV3A (28202071, 24 kDa) 141 Glucosamine-6-phosphate isomerase 6 (23%) 324 (94375862, 29 kDa) splicing factor, arginine/serine-rich 1 6 (29%) 315 (34328400, 28 kDa) otubain 1 (19527388, 23%) 5 (23%) 309 cytoskeleton-associated protein 1 (93277119, 5 (20%) 283 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 4 (21%) 253 monooxygenase activation protein (13928824, 29 kDa) DNA fragmentation factor, alpha subunit 2 (7%) 213 isoform a (70608119, 37 kDa) RIKEN cDNA 0610040D20 (12963537, 27 2 (16%) 212 kDa) 4 (14%) 198 phosphopantothenoylcysteine synthetase Report 1818 Vijay (76096364, 34 kDa) Eukaryotic translation initiation factor 3 subunit 2 (8%) 181 1 (109468723, 29 kDa) spermidine synthase (6678131, 34 kDa) 2 (11%) 179 tubulin, beta, 2 (22165384, 50 kDa) 4 (11%) 144 translin-associatedpage 5factor X (8394490, 33 kDa) 3 (15%) 140 inorganic pyrophosphatase 2 (22203753, 39 2 (7%) 139 kDa) aldo-keto reductase family 1, member E1 5 (18%) 120 (93277108, 35 kDa) protein phosphatase 1, regulatory (inhibitor) 2 (17%) 108 subunit 2 (18859587, 23 kDa) 2 cDEAE Ts SB12-94-6 tyrosine 3-monooxygenase/tryptophan 5- 17 (64%) 2257 band 2 monooxygenase activation protein (13928824, 29 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (30%) 650 monooxygenase activation protein, zeta polypeptide (62990183, 28 kDa) tropomyosin 3, gamma isoform 1 (52353308, 1087 29 kDa) tropomyosin 3, gamma (40254525, 33 kDa) 13 (44%) 721 EF hand domain containing 2 (31981086, 27 12 (46%) 644 kDa) Glyoxalase domain containing 4 (21313080, 34 10 (43%) 592 kDa) chloride intracellular channel 1 (15617203, 27 6 (31%) 496 kDa) splicing factor, arginine/serine-rich 2 (6755478, 5 (21%) 473 25 kDa) chromatin modifying protein 5 (13386442, 25 5 (29%) 369 kDa) cytokine induced protein 29 kDa (13384730, 24 8 (42%) 363 kDa) protein FAM49B (21450053, 37 kDa) 3 (12%) 333 proteasome 26S non-ATPase subunit 9 4 (22%) 323 (18426862, 25 kDa) acidic (leucine-rich) nuclear phosphoprotein 32 3 (5%) 312 family, member E (31542131, 30 kDa) carnitine deficiency-associated gene expressed 3 (36%) 299 in ventricle 3 isoform CDV3A (28202071, 24 kDa) coiled-coil domain containing 104 (30352008, 6 (26%) 286 40 kDa) Glucosamine-6-phosphate isomerase 4 (24%) 283 (94375862, 29 kDa) phosphoglycolate phosphatase (40254507, 35 4 (14%) 280 kDa)

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Eukaryotic translation initiation factor 3 subunit 2 (8%) 181 1 (109468723, 29 kDa) spermidine synthase (6678131, 34 kDa) 2 (11%) 179 tubulin, beta, 2 (22165384, 50 kDa) 4 (11%) 144 translin-associated factor X (8394490, 33 kDa) 3 (15%) 140 inorganic pyrophosphatase 2 (22203753, 39 2 (7%) 139 kDa) aldo-keto reductase family 1, member E1 5 (18%) 120 (93277108, 35 kDa) protein phosphatase 1, regulatory (inhibitor) 2 (17%) 108 subunit 2 (18859587, 23 kDa) 2 cDEAE Ts SB12-94-6 tyrosine 3-monooxygenase/tryptophan 5- 17 (64%) 2257 band 2 monooxygenase activation protein (13928824, 29 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (30%) 650 monooxygenase activation protein, zeta polypeptide (62990183, 28 kDa) tropomyosin 3, gamma isoform 1 (52353308, 1087 29 kDa) tropomyosin 3, gamma (40254525, 33 kDa) 13 (44%) 721 EF hand domain containing 2 (31981086, 27 12 (46%) 644 kDa) Glyoxalase domain containing 4 (21313080, 34 10 (43%) 592 kDa) chloride intracellular channel 1 (15617203, 27 6 (31%) 496 kDa) splicing factor, arginine/serine-rich 2 (6755478, 5 (21%)142 473 25 kDa) chromatin modifying protein 5 (13386442, 25 5 (29%) 369 kDa) cytokine induced protein 29 kDa (13384730, 24 8 (42%) 363 kDa) protein FAM49B (21450053, 37 kDa) 3 (12%) 333 proteasome 26S non-ATPase subunit 9 4 (22%) 323 (18426862, 25 kDa) acidic (leucine-rich) nuclear phosphoprotein 32 3 (5%) 312 family, member E (31542131, 30 kDa) carnitine deficiency-associated gene expressed 3 (36%) 299 in ventricle 3 isoform CDV3A (28202071, 24 kDa) coiled-coil domain containing 104 (30352008, 6 (26%) 286 40 kDa) Glucosamine-6-phosphate isomerase 4 (24%) 283 (94375862, 29 kDa) phosphoglycolate phosphatase (40254507, 35 4 (14%) 280 kDa) Report 1818 Vijay

protein phosphatase 1, regulatory (inhibitor) 4 (22%) 271 subunit 2 (18859587, 23 kDa) page 6 annexin A5 (6753060, 36 kDa) 5 (18%) 252 phosphatidylethanolamine binding protein 1 3 (28%) 225 (84794552, 21 kDa) splicing factor, arginine/serine-rich 1 5 (22%) 224 (34328400, 28 kDa) cytoskeleton-associated protein 1 (93277119, 4 (18%) 195 28 kDa) ribosomal protein S3 (6755372, 27 kDa) 5 (24%) 163 acidic nuclear phosphoprotein 32 family, 4 (17%) 157 member B (18700032, 31 kDa) splicing factor, arginine/serine-rich 7 5 (36%) 146 (84781668, 18 kDa) tubulin, beta, 2 (22165384, 50 kDa) 3 (8%) 140 3 cDEAE Ts SB12-94-7 tyrosine 3-monooxygenase/tryptophan 5- 13 (56%) 3230 band 3 monooxygenase activation protein, zeta polypeptide (62990183, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 15 (59%) 2192 monooxygenase activation protein, theta polypeptide (6756039, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 11 (43%) 1221 monooxygenase activation protein, beta polypeptide (31543974, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 7 (34%) 961 monooxygenase activation protein (13928824, 29 kDa) Ubiquitin carboxyl-terminal hydrolase isozyme 5 (34%) 563 (34874586, 26 kDa) inositol (myo)-1(or 4)-monophosphatase 1 10 (32%) 419 (31980942, 31 kDa) Ran-specific GTPase-activating protein 5 (31%) 389 (62658198, 24 kDa) Xlr-related, meiosis regulated isoform 1 4 (41%) 385 (51772621, 18 kDa) chloride intracellular channel 4 (7304963, 29 5 (30%) 367 kDa) ankyrin repeat and SOCS box-containing 4 (12%) 359 protein 9 (21311897, 32 kDa) proteasome 26S non-ATPase subunit 9 4 (18%) 331 (13385506, 28 kDa) platelet-activating factor acetylhydrolase alpha 4 (19%) 324 2 subunit (11693154, 26 kDa) phosphatidylethanolamine binding protein 1 3 (28%) 298 (84794552, 21 kDa)

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protein phosphatase 1, regulatory (inhibitor) 4 (22%) 271 subunit 2 (18859587, 23 kDa) annexin A5 (6753060, 36 kDa) 5 (18%) 252 phosphatidylethanolamine binding protein 1 3 (28%) 225 (84794552, 21 kDa) splicing factor, arginine/serine-rich 1 5 (22%) 224 (34328400, 28 kDa) cytoskeleton-associated protein 1 (93277119, 4 (18%) 195 28 kDa) ribosomal protein S3 (6755372, 27 kDa) 5 (24%) 163 acidic nuclear phosphoprotein 32 family, 4 (17%) 157 member B (18700032, 31 kDa) splicing factor, arginine/serine-rich 7 5 (36%) 146 (84781668, 18 kDa) tubulin, beta, 2 (22165384, 50 kDa) 3 (8%) 140 3 cDEAE Ts SB12-94-7 tyrosine 3-monooxygenase/tryptophan 5- 13 (56%) 3230 band 3 monooxygenase activation protein, zeta polypeptide (62990183, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 15 (59%) 2192 monooxygenase activation protein, theta polypeptide (6756039, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 11 (43%) 1221 monooxygenase activation protein, beta polypeptide (31543974, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 7 (34%) 961 monooxygenase activation protein (13928824, 143 29 kDa) Ubiquitin carboxyl-terminal hydrolase isozyme 5 (34%) 563 (34874586, 26 kDa) inositol (myo)-1(or 4)-monophosphatase 1 10 (32%) 419 (31980942, 31 kDa) Ran-specific GTPase-activating protein 5 (31%) 389 (62658198, 24 kDa) Xlr-related, meiosis regulated isoform 1 4 (41%) 385 (51772621, 18 kDa) chloride intracellular channel 4 (7304963, 29 5 (30%) 367 kDa) ankyrin repeat and SOCS box-containing 4 (12%) 359 protein 9 (21311897, 32 kDa) proteasome 26S non-ATPase subunit 9 4 (18%) 331 (13385506, 28 kDa) platelet-activating factor acetylhydrolase alpha 4 (19%) 324 2 subunit (11693154, 26 kDa) phosphatidylethanolamine binding protein 1 3 (28%) 298 (84794552, 21 kDa) Report 1818 Vijay

glutamate-cysteine ligase , modifier subunit 3 (13%) 295 (6680019, 31 kDa) Phosphoglyceratepage mutase7 1 (94369185, 29 kDa) 5 (28%) 233 Bcl2-associated athanogene 1 isoform 1L 4 (14%) 221 (34398362, 40 kDa) heme binding protein 2 (9507129, 23 kDa) 4 (18%) 219 calcyclin binding protein (33468885, 35 kDa) 5 (35%) 209 chloride intracellular channel 1 (15617203, 27 2 (12%) 161 kDa) acidic (leucine-rich) nuclear phosphoprotein 32 5 (15%) 155 family, member A (6978499, 29 kDa) protein phosphatase 1, regulatory (inhibitor) 2 (17%) 151 subunit 2 (18859587, 23 kDa) purine-nucleoside phosphorylase (7305395, 33 2 (10%) 144 kDa) splicing factor, arginine/serine-rich 7 3 (20%) 141 (84781668, 18 kDa) cytochrome b5 reductase b5R.2 (28893247, 31 3 (11%) 114 kDa( methylthioadenosine phosphorylase (45544618, 2 (9%) 110 31 kDa) 4 DEAE Sp SB12-94-8 lactate dehydrogenase 3, C chain, sperm 14 (44%) 1651 band 1 specific (7305229, 36 kDa) pyruvate dehydrogenase (lipoamide) beta 11 (37%) 1383 (18152793, 39 kDa) annexin A5 (6753060, 36 kDa) 9 (28%) 591 tyrosine 3-monooxygenase/tryptophan 5- 8 (30%) 486 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 3 (13%) 135 monooxygenase activation protein (13928824, 29 kDa) phosphoribosyl pyrophosphate synthetase 1 6 (22%) 335 (8394053, 35 kDa) ATPase, H+ transporting, V1 subunit D 2 (10%) 308 (12963799, 28 kDa) annexin A13 (23956196, 36 kDa) 5 (21%) 307 Rhabdoid tumor deletion region protein 1 5 (16%) 301 isoform 2 (94389386, 38 kDa) ATP synthase, H+ transporting mitochondrial 7 (18%) 292 F1 complex, beta subunit (31980648, 56 kDa) N-terminal fragment STIP1 homology and U-box containing protein 5 (22%) 292 1 (9789907, 35 kDa) succinate-CoA ligase, GDP-forming, alpha 2 (8%) 265 subunit (9845299, 35 kDa)

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glutamate-cysteine ligase , modifier subunit 3 (13%) 295 (6680019, 31 kDa) Phosphoglycerate mutase 1 (94369185, 29 kDa) 5 (28%) 233 Bcl2-associated athanogene 1 isoform 1L 4 (14%) 221 (34398362, 40 kDa) heme binding protein 2 (9507129, 23 kDa) 4 (18%) 219 calcyclin binding protein (33468885, 35 kDa) 5 (35%) 209 chloride intracellular channel 1 (15617203, 27 2 (12%) 161 kDa) acidic (leucine-rich) nuclear phosphoprotein 32 5 (15%) 155 family, member A (6978499, 29 kDa) protein phosphatase 1, regulatory (inhibitor) 2 (17%) 151 subunit 2 (18859587, 23 kDa) purine-nucleoside phosphorylase (7305395, 33 2 (10%) 144 kDa) splicing factor, arginine/serine-rich 7 3 (20%) 141 (84781668, 18 kDa) cytochrome b5 reductase b5R.2 (28893247, 31 3 (11%) 114 kDa( methylthioadenosine phosphorylase (45544618, 2 (9%) 110 31 kDa) 4 DEAE Sp SB12-94-8 lactate dehydrogenase 3, C chain, sperm 14 (44%) 1651 band 1 specific (7305229, 36 kDa) pyruvate dehydrogenase (lipoamide) beta 11 (37%) 1383 (18152793, 39 kDa) annexin A5 (6753060, 36 kDa) 9 (28%) 591 tyrosine 3-monooxygenase/tryptophan 5- 8 (30%)144 486 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 3 (13%) 135 monooxygenase activation protein (13928824, 29 kDa) phosphoribosyl pyrophosphate synthetase 1 6 (22%) 335 (8394053, 35 kDa) ATPase, H+ transporting, V1 subunit D 2 (10%) 308 (12963799, 28 kDa) annexin A13 (23956196, 36 kDa) 5 (21%) 307 Rhabdoid tumor deletion region protein 1 5 (16%) 301 isoform 2 (94389386, 38 kDa) ATP synthase, H+ transporting mitochondrial 7 (18%) 292 F1 complex, beta subunit (31980648, 56 kDa) N-terminal fragment STIP1 homology and U-box containing protein 5 (22%) 292 1 (9789907, 35 kDa) succinate-CoA ligase, GDP-forming, alpha 2 (8%) 265 subunit (9845299, 35 kDa) Report 1818 Vijay

protein phosphatase 1, catalytic subunit, beta 3 (13%) 254 (6981388, 38page kDa) 8 protein phosphatase 1, catalytic subunit, gamma 3 (13%) 233 isoform (11968062, 38 kDa) Glucosamine-6-phosphate isomerase 6 (24%) 249 (109507234, 33 kDa) protein phosphatase 1, regulatory (inhibitor) 4 (14%) 223 subunit 2 (63701773, 35 kDa) tubulin, beta, 2 (22165384, 50 kDa) 7 (19%) 220 heat shock 70kDa protein 4 like (40254361, 9 10 (14%) 219 kDa)C-terminal fragment radial spoke head protein 9 homolog 7 (22%) 218 (21312974, 31 kDa) tubulin, alpha 8 (8394493, 51 kDa) 2 (7%) 214 deoxyribose-phosphate aldolase-like 5 (21%) 186 (27777677, 36 kDa) hypothetical protein LOC67078 (40254507, 35 3 (11%) 179 kDa) N-ethylmaleimide sensitive fusion protein 3 (12%) 175 attachment protein alpha (13385392, 33 kDa) protein phosphatase 6, catalytic subunit 3 (11%) 161 (21312758, 36 kDa) nascent polypeptide-associated complex alpha 2 (14%) 143 polypeptide (94371769, 22 kDa) coenzyme Q9 homolog (33859690, 35 kDa) 2 (8%) 142 protein phosphatase 2a, catalytic subunit, beta 2 (8%) 134 isoform (8394021, 36 kDa) Charged multivesicular body protein 4b 4 (17%) 133 (109471140, 29 kDa) thioredoxin-like (18266686, 33 kDa) 3 (15%) 129 carbonic anhydrase 4 (6671678, 35 kDa) 3 (11%) 110 5 DEAE Sp SB12-94-9 tropomyosin 3, gamma isoform 2 (29336093, 8 (57%) 1175 band 2 29 kDa) pyruvate dehydrogenase (lipoamide) beta 7 (27%) 1170 (18152793, 39 kDa) glucosamine-6-phosphate deaminase 2 11 (46%) 1007 (83999999, 31 kDa) Glucosamine-6-phosphate isomerase 6 (29%) 334 (109507234, 33 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (35%) 848 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 7 (39%) 490 monooxygenase activation protein (13928824, 29 kDa)

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protein phosphatase 1, catalytic subunit, beta 3 (13%) 254 (6981388, 38 kDa) protein phosphatase 1, catalytic subunit, gamma 3 (13%) 233 isoform (11968062, 38 kDa) Glucosamine-6-phosphate isomerase 6 (24%) 249 (109507234, 33 kDa) protein phosphatase 1, regulatory (inhibitor) 4 (14%) 223 subunit 2 (63701773, 35 kDa) tubulin, beta, 2 (22165384, 50 kDa) 7 (19%) 220 heat shock 70kDa protein 4 like (40254361, 9 10 (14%) 219 kDa)C-terminal fragment radial spoke head protein 9 homolog 7 (22%) 218 (21312974, 31 kDa) tubulin, alpha 8 (8394493, 51 kDa) 2 (7%) 214 deoxyribose-phosphate aldolase-like 5 (21%) 186 (27777677, 36 kDa) hypothetical protein LOC67078 (40254507, 35 3 (11%) 179 kDa) N-ethylmaleimide sensitive fusion protein 3 (12%) 175 attachment protein alpha (13385392, 33 kDa) protein phosphatase 6, catalytic subunit 3 (11%) 161 (21312758, 36 kDa) nascent polypeptide-associated complex alpha 2 (14%) 143 polypeptide (94371769, 22 kDa) coenzyme Q9 homolog (33859690, 35 kDa) 2 (8%) 142 protein phosphatase 2a, catalytic subunit, beta 2 (8%)145 134 isoform (8394021, 36 kDa) Charged multivesicular body protein 4b 4 (17%) 133 (109471140, 29 kDa) thioredoxin-like (18266686, 33 kDa) 3 (15%) 129 carbonic anhydrase 4 (6671678, 35 kDa) 3 (11%) 110 5 DEAE Sp SB12-94-9 tropomyosin 3, gamma isoform 2 (29336093, 8 (57%) 1175 band 2 29 kDa) pyruvate dehydrogenase (lipoamide) beta 7 (27%) 1170 (18152793, 39 kDa) glucosamine-6-phosphate deaminase 2 11 (46%) 1007 (83999999, 31 kDa) Glucosamine-6-phosphate isomerase 6 (29%) 334 (109507234, 33 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (35%) 848 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 7 (39%) 490 monooxygenase activation protein (13928824, Report 1818 Vijay 29 kDa) proteasome (prosome, macropain) subunit, 11 (57%) 608 alpha type 1 (33563282, 30 kDa) complement component 1, q subcomponent 4 (22%) 580 binding protein (112181167, 31 kDa) page 9 lactate dehydrogenase 3, C chain, sperm 8 (31%) 568 specific (7305229, 36 kDa) ATPase, H+ transporting, V1 subunit D 4 (16%) 428 (12963799, 28 kDa) radial spoke head protein 9 homolog 7 (25%) 415 (21312974, 31 kDa) ATP synthase gamma chain, mitochondrial 7 (16%) 365 precursor (94366099, 45 kDa) chloride intracellular channel 1 (15617203, 27 4 (21%) 278 kDa) purine-nucleoside phosphorylase (7305395, 33 3 (13%) 212 kDa) proteasome activator subunit 2 isoform 1 2 (10%) 168 (20137004, 27 kDa) ATPase, H+ transporting, V1 subunit E-like 2 5 (23%) 147 isoform 2 (42475954, 26 kDa) N-ethylmaleimide sensitive fusion protein 2 (8%) 143 attachment protein alpha (13385392, 34 kDa) diaphorase 1 (19745150, 34 kDa) 4 (13%) 142 zona pellucida binding protein (7657605, 46 2 (4%) 140 kDa) tropomyosin 4 (47894398, 29 kDa) 3 (15%) 124 heat shock 70kDa protein 4 like (40254361, 95 6 (8%) 122 kDa) C-terminal fragment deoxyribose-phosphate aldolase-like 3 (13%) 120 (27777677, 35 kDa) annexin A5 (6753060, 36 kDa) 3 (10%) 116 protein phosphatase 1, regulatory (inhibitor) 2 (10%) 109 subunit 2 (18859587, 23 kDa) 6 DEAE Sp SB12-94-10 tyrosine 3-monooxygenase/tryptophan 5- 15 (66%) 2881 band 3 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) proteasome (prosome, macropain) subunit, 7 (34%) 1410 alpha type 4 (8394069, 30 kDa) proacrosin binding protein (8392845, 62 kDa) 6 (14%) 1322 C-terminal fragment tyrosine 3-monooxygenase/tryptophan 5- 11 (46%) 932 monooxygenase activation protein, beta polypeptide (31543974, 28 kDa) proteasome (prosome, macropain) subunit, 5 (29%) 632 alpha type 5 (7106387, 27 kDa)

page 10 Report 1818 Vijay

proteasome (prosome, macropain) subunit, 11 (57%) 608 alpha type 1 (33563282, 30 kDa) complement component 1, q subcomponent 4 (22%) 580 binding protein (112181167, 31 kDa) lactate dehydrogenase 3, C chain, sperm 8 (31%) 568 specific (7305229, 36 kDa) ATPase, H+ transporting, V1 subunit D 4 (16%) 428 (12963799, 28 kDa) radial spoke head protein 9 homolog 7 (25%) 415 (21312974, 31 kDa) ATP synthase gamma chain, mitochondrial 7 (16%) 365 precursor (94366099, 45 kDa) chloride intracellular channel 1 (15617203, 27 4 (21%) 278 kDa) purine-nucleoside phosphorylase (7305395, 33 3 (13%) 212 kDa) proteasome activator subunit 2 isoform 1 2 (10%) 168 (20137004, 27 kDa) ATPase, H+ transporting, V1 subunit E-like 2 5 (23%) 147 isoform 2 (42475954, 26 kDa) N-ethylmaleimide sensitive fusion protein 2 (8%) 143 attachment protein alpha (13385392, 34 kDa) diaphorase 1 (19745150, 34 kDa) 4 (13%) 142 zona pellucida binding protein (7657605, 46 2 (4%) 140 kDa) tropomyosin 4 (47894398, 29 kDa) 3 (15%)146 124 heat shock 70kDa protein 4 like (40254361, 95 6 (8%) 122 kDa) C-terminal fragment deoxyribose-phosphate aldolase-like 3 (13%) 120 (27777677, 35 kDa) annexin A5 (6753060, 36 kDa) 3 (10%) 116 protein phosphatase 1, regulatory (inhibitor) 2 (10%) 109 subunit 2 (18859587, 23 kDa) 6 DEAE Sp SB12-94-10 tyrosine 3-monooxygenase/tryptophan 5- 15 (66%) 2881 band 3 monooxygenase activation protein, zeta polypeptide (6756041, 28 kDa) proteasome (prosome, macropain) subunit, 7 (34%) 1410 alpha type 4 (8394069, 30 kDa) proacrosin binding protein (8392845, 62 kDa) 6 (14%) 1322 C-terminal fragment tyrosine 3-monooxygenase/tryptophan 5- 11 (46%) 932 monooxygenase activation protein, beta polypeptide (31543974, 28 kDa) proteasome (prosome, macropain) subunit, Report5 (29%) 1818 Vijay 632 alpha type 5 (7106387, 27 kDa) proteasome (prosome, macropain) subunit, 8 (32%) 619 alpha type 3 (8394066, 29 kDa) proteasome (prosome, macropain) subunit, 9 (46%) 614 alpha type, 8 isoform 1 (38083795, 28 KDa) page 10 tyrosine 3-monooxygenase/tryptophan 5- 10 (35%) 585 monooxygenase activation protein, theta polypeptide (6756039, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (32%) 417 monooxygenase activation protein, eta (6756037, 28 kDa) purine-nucleoside phosphorylase (7305395, 33 5 (17%) 414 kDa) 3-monooxgenase/tryptophan 5-monooxygenase 4 (30%) 413 activation protein, gamma polypeptide (31543976, 28 kDa) ubiquitin carboxyl-terminal esterase L3 6 (32%) 377 (7710106, 26 kDa) glutamate-cysteine ligase , modifier subunit 2 (7%) 285 (6680019, 31 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (23%) 284 monooxygenase activation protein (13928824, 29 kDa) Bcl2-associated athanogene 1 isoform 1L 2 (12%) 227 (34398362, 40 kDa) solute carrier family 25, member 5 (22094075, 4 (14%) 220 33 kDa) reticulocalbin 1 (6677691 ,38 kDa) 2 (3%) 182 U2 small nuclear ribonucleoprotein polypeptide 4 (19%) 162 A' (31981942, 28 kDa) tropomyosin 3, gamma isoform 2 (29336093, 4 (13%) 152 29 kDa) chloride intracellular channel 1 (15617203, 27 2 (9%) 121 kDa) tropomyosin 1, alpha isoform f (14192925, 29 3 (14%) 79 kDa)

page 11 Report 1818 Vijay

proteasome (prosome, macropain) subunit, 8 (32%) 619 alpha type 3 (8394066, 29 kDa) proteasome (prosome, macropain) subunit, 9 (46%) 614 alpha type, 8 isoform 1 (38083795, 28 KDa) tyrosine 3-monooxygenase/tryptophan 5- 10 (35%) 585 monooxygenase activation protein, theta polypeptide (6756039, 28 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (32%) 417 monooxygenase activation protein, eta (6756037, 28 kDa) purine-nucleoside phosphorylase (7305395, 33 5 (17%) 414 kDa) 3-monooxgenase/tryptophan 5-monooxygenase 4 (30%) 413 activation protein, gamma polypeptide (31543976, 28 kDa) ubiquitin carboxyl-terminal esterase L3 6 (32%) 377 (7710106, 26 kDa) glutamate-cysteine ligase , modifier subunit 2 (7%) 285 (6680019, 31 kDa) tyrosine 3-monooxygenase/tryptophan 5- 5 (23%) 284 monooxygenase activation protein (13928824, 29 kDa) Bcl2-associated athanogene 1 isoform 1L 2 (12%) 227 (34398362, 40 kDa) solute carrier family 25, member 5 (22094075, 4 (14%) 220 33 kDa) 147 reticulocalbin 1 (6677691 ,38 kDa) 2 (3%) 182 U2 small nuclear ribonucleoprotein polypeptide 4 (19%) 162 A' (31981942, 28 kDa) tropomyosin 3, gamma isoform 2 (29336093, 4 (13%) 152 29 kDa) chloride intracellular channel 1 (15617203, 27 2 (9%) 121 kDa) tropomyosin 1, alpha isoform f (14192925, 29 3 (14%) 79 kDa)

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SB12-91-20; Band 87 Three proteins were identified in band 87 and include endoplasmic reticulum protein ERp29 precursor, hydroxyprostaglandin dehydrogenase 15, and cutC copper transporter homolog. The main component of this band is endoplasmic reticulum protein ERp29.

SB12-91-21; Band 30 A total of 8 proteins were identified in band 30. The major component is glyceraldehyde-3- phosphate dehydrogenase spermatogenic (also identified in bands 13 and 82) which was identified by 17 peptides covering 52% of the protein sequence. Additional proteins identified in these bands include ubiquinol cytochrome c reductase core protein 2, acetyl-Coenzyme A acetyltransferase 1 precursor, lactate dehydrogenase 3 C chain sperm specific, glutathione peroxidase 4 isoform 1 precursor, acetyl-Coenzyme A acyltransferase 2, A-kinase anchor protein 4 isoform a, and Aminomethyltransferase.

Conclusions APPENDIX II Almost all of the 2D gel bands analyzed in these experiments were found to contain multiple proteins. The investigator should note that we typically associate the difference in degree of staining to the most abundant components of a band, however, it is common practice to perform follow up experimentsProteins in order Identified to verify the By differential Microsequencing expression ofOf these 2D- proteins.DIGE Spots

Table 2. Summary of results using rodent protein database. Digest Identification Peptides # Mascot Sample sample (NCBI accession number, calculated MW, calculated pI) (sequence Score Name number coverage) 1 Vijay SB12- outer dense fiber of sperm tails 2 (7305339, 74.08 kDa, 19 (35%) 5088 2D 91-1 7.2) band 5 acyl-CoA synthetase long-chain family member 1 17 (32%) 2713 (31560705, 78.93 kDa, 6.8) A-kinase anchor protein 4 isoform a (110347481, 95.56 10 (19%) 631 kDa, 6.6) prostaglandin-endoperoxide synthase 2 (31981525, 5 (12%) 378 69.71 kDa, 7.0) methylcrotonoyl-Coenzyme A carboxylase 1 (alpha) 5 (9%) 226 (31980706, 79.67 kDa, 7.7) 2 Vijay SB12- mitochondrial trifunctional protein, alpha subunit 22 (30%) 5557 2D 91-2 (33859811, 83.30 kDa, 9.2) band 6 Lactotransferrin (31560677, 79.67 kDa, 8.9) 30 (50%) 3804 hydroxysteroid (17-beta) dehydrogenase 4 (31982273, 4 (6%) 328 79.95 kDa, 8.8) ubiquinol cytochrome c reductase core protein 2 3 (11%) 176 (22267442, 48.26 kDa, 9.3) L-specific multifunctional beta-oxdiation protein Report 18542 (3%) Vijay/Ramdas 122 (31541815, 78.82 kDa, 9.2) citrate synthase-like protein (13386272, 52.61 kDa, 8.9) 2 (5%) 98 3 Vijay SB12- carnitine acetyltransferase (85662408, 71.25 kDa, 8.6) 20 (41%) 2978 2D 91-3 pyruvate kinase 3 (31981562, 58.38 kDa, 7.2) 16 (40%) 1196 band 13 glyceraldehyde-3-phosphatepage dehydrogenase, 6 8 (23%) 991 spermatogenic (6679939, 48.10 kDa, 8.2) A-kinase anchor protein 4 isoform a (110347481, 95.56 6 (11%) 704 kDa, 6.6) PREDICTED: similar to Proline oxidase, mitochondrial 5 (12%) 302 precursor (Proline dehydrogenase) (94401086, 76.40 kDa, 9.0) Calicin (50582595, 67.73 kDa, 8.5) 4 (10%) 186 4 Vijay SB12- outer dense fiber of sperm tails 2 (7305339, 74.08 kDa, 20 (30%) 4786 2D 91-4 7.2) C-terminal fragment band 16 PREDICTED: similar to Succinyl-CoA ligase [GDP- 8 (23%) 867 forming] beta-chain, mitochondrial precursor (94378914, 52.31 kDa, 5.4) SPFH domain family, member 2 (23956396, 38.08 kDa, 4 (14%) 471 5.4) succinate-Coenzyme A ligase, ADP-forming, beta 5 (14%) 326 subunit (46849708, 50.42 kDa, 6.6) COP9 signalosome148 subunit 4 (6753490, 46.54 kDa, 5.6) 3 (8%) 257 Vijay fructose bisphosphatase 1 (9506589, 37.29 kDa, 6.2) 2 (7%) 357 SB12- 5 2D 91-5 band 18 Vijay fructose bisphosphatase 1 (9506589, 37.29 kDa, 6.2) 14 (46%) 3190 SB12- 6 2D aldo-keto reductase family 1, member B7 (6753148, 2 (6%) 54 91-6 band 20 36.12 kDa, 6.5) 7 Vijay SB12- phosphoglycerate kinase 2 (13654249, 12.96 kDa, 6.0) 21 (72%) 12959 2D 91-7 ARP1 actin-related protein 1 homolog B (22122615, 6 (27%) 608 band 21 42.37 kDa, 6.0) translocase of inner mitochondrial membrane 44 2 (5%) 441 homolog (8394449, 51.37 kDa, 8.3) tripeptidyl peptidase I (6753448, 61.76 kDa, 6.1) 2 (4%) 364 S-adenosylhomocysteine hydrolase (61098092, 48.16 5 (16%) 353 kDa, 6.0) actin, beta, cytoplasmic (6671509, 42.05 kDa, 5.3) 2 (12%) 282 N-acetyl galactosaminidase, alpha (31560666, 47.70 2 (5%) 141 kDa, 6.0) 8 Vijay SB12- alpha enolase (70794816, 47.45 kDa, 6.4) 26 (70%) 10255 2D 91-8 eukaryotic translation elongation factor 1 gamma 13 (30%) 1133 band 22 (110625979, 50.37 kDa, 6.3) aldolase 1, A isoform (6671539, 39.79 kDa, 8.3) 3 (14%) 532 3-oxoacyl-ACP synthase, mitochondrial (58037235, 9 (27%) 516 49.22 kDa, 6.6) DEAD (Asp-Glu-Ala-Asp) box polypeptide 48 7 (30%) 480 (20149756, 47.10 kDa, 6.3)

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3 Vijay SB12- carnitine acetyltransferase (85662408, 71.25 kDa, 8.6) 20 (41%) 2978 2D 91-3 pyruvate kinase 3 (31981562, 58.38 kDa, 7.2) 16 (40%) 1196 band 13 glyceraldehyde-3-phosphate dehydrogenase, 8 (23%) 991 spermatogenic (6679939, 48.10 kDa, 8.2) A-kinase anchor protein 4 isoform a (110347481, 95.56 6 (11%)149 704 kDa, 6.6) PREDICTED: similar to Proline oxidase, mitochondrial 5 (12%) 302 precursor (Proline dehydrogenase) (94401086, 76.40 kDa, 9.0) Calicin (50582595, 67.73 kDa, 8.5) 4 (10%) 186 4 Vijay SB12- outer dense fiber of sperm tails 2 (7305339, 74.08 kDa, 20 (30%) 4786 2D 91-4 7.2) C-terminal fragment band 16 PREDICTED: similar to Succinyl-CoA ligase [GDP- 8 (23%) 867 forming] beta-chain, mitochondrial precursor (94378914, 52.31 kDa, 5.4) SPFH domain family, member 2 (23956396, 38.08 kDa, 4 (14%) 471 5.4) succinate-Coenzyme A ligase, ADP-forming, beta 5 (14%) 326 subunit (46849708, 50.42 kDa, 6.6) COP9 signalosome subunit 4 (6753490, 46.54 kDa, 5.6) 3 (8%) 257 Vijay fructose bisphosphatase 1 (9506589, 37.29 kDa, 6.2) 2 (7%) 357 SB12- 5 2D 91-5 band 18 Vijay fructose bisphosphatase 1 (9506589, 37.29 kDa, 6.2) 14 (46%) 3190 SB12- 6 2D aldo-keto reductase family 1, member B7 (6753148, 2 (6%) 54 91-6 band 20 36.12 kDa, 6.5) 7 Vijay SB12- phosphoglycerate kinase 2 (13654249, 12.96 kDa, 6.0) 21 (72%) 12959 2D 91-7 ARP1 actin-related protein 1 homolog B (22122615, 6 (27%) 608 band 21 42.37 kDa, 6.0) translocase of inner mitochondrial membrane 44 2 (5%) 441 homolog (8394449, 51.37 kDa, 8.3) tripeptidyl peptidase I (6753448, 61.76 kDa, 6.1) 2 (4%) 364 S-adenosylhomocysteine hydrolase (61098092, 48.16 5 (16%) 353 kDa, 6.0) actin, beta, cytoplasmic (6671509, 42.05 kDa, 5.3) 2 (12%) 282 N-acetyl galactosaminidase, alpha (31560666, 47.70 2 (5%) 141 kDa, 6.0) 8 Vijay SB12- alpha enolase (70794816, 47.45 kDa, 6.4) 26 (70%) 10255 2D 91-8 eukaryotic translation elongation factor 1 gamma 13 (30%) 1133 band 22 (110625979, 50.37 kDa, 6.3) aldolase 1, A isoform (6671539, 39.79 kDa, 8.3) 3 (14%) 532 3-oxoacyl-ACP synthase, mitochondrial (58037235, 9 (27%) 516 49.22 kDa, 6.6) DEAD (Asp-Glu-Ala-Asp) box polypeptide 48 7 (30%) 480 (20149756, 47.10 kDa, 6.3)

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phosphoglycerate kinase 2 (13654249, 45.25 kDa, 6.4) 3 (11%) 206 phosphoglycerateproliferation-associated kinase 2G 2 (413654249, (6755100, 45.25 44.01 kDa, kDa, 6.4) 6.4) 35 (11%)(15%) 206118 9 Vijay SB12- proliferation-associatedphosphoglycerate kinase 2G 2 (413654249, (6755100, 45.25 44.01 kDa, kDa, 6.4) 6.4) 30 5 (15%) (87%) 14993 118 9 Vijay2D SB12-91-9 phosphoglyceratetranslocator of inner kinase mitochondrial 2 (13654249, membrane 45.25 kDa, 44 6.4) 308 (27%)(87%) 14993 654 band 23 2D 91-9 translocator(19705563, 51.49of inner kDa, mitochondrial 8.5) membrane 44 8 (27%) 654 band 23 (19705563,ARP1 actin-related 51.49 kDa, protein 8.5) 1 homolog A (8392847, 4 (17%) 544 ARP142.70 kDa,actin-related 6.2) protein 1 homolog A (8392847, 4 (17%) 544 42.70actin, beta,kDa, cytoplasmic6.2) (6671509, 42.05 kDa, 5.3) 4 (17%) 344 actin,mitochondrial beta, cytoplasmic acyl-CoA (6671509, thioesterase 42.05 1 (40538846, kDa, 5.3) 4 (17%)(11%) 344237 mitochondrial49.84 kDa, 7.2) acyl-CoA thioesterase 1 (40538846, 4 (11%) 237 49.84S-adenosylhomocysteinase kDa, 7.2) (262263372, 47.69 kDa, 6.1) 2 (5%) 187 S-adenosylhomocysteinaseNAD kinase domain-containing (262263372, protein 1 47.69 isoform kDa, 1 6.1) 5 2 (14%)(5%) 187 96 NAD (146134392, kinase domain-containing 48.56 kDa, 7.1) protein 1 isoform 1 5 (14%) 96 10 Vijay SB12- sorbitol(146134392, dehydrogenase 48.56 kDa, 1 7.1)(22128627, 38.80 kDa, 6.6) 20 (62%) 5852 10 Vijay2D SB12-91-10 sorbitolphosphoglycerate dehydrogenase kinase 1 2(22128627, (13654249, 38.80 45.25 kDa, kDa, 6.6) 6.4) 2012 (62%)(41%) 5852 705 band 34 2D 91-10 phosphoglycerateglutathione S-transferas kinasee, 2 mu (13654249, 5 (6754086, 45.25 27.02 kDa, kDa, 6.4) 126 (28%)(41%) 705463 band 34 glutathione6.8) S-transferase, mu 5 (6754086, 27.02 kDa, 6 (28%) 463 6.8)alcohol dehydrogenase 5 (class III), chi polypeptide 7 (21%) 429 alcohol(31982511, dehydrogenase 40.32 kDa, 57.0) (class III), chi polypeptide 7 (21%) 429 (31982511,heterogeneous 40.32 nuclear kDa, ribonucleoprotein 7.0) C (8393544, 3 (12%) 363 heterogeneous34.42 kDa, 4.9) nuclear ribonucleoprotein C (8393544, 3 (12%) 363 34.42aldolase kDa, 1, A4.9) isoform (6671539, 39.79 kDa, 8.3) 2 (6%) 284 aldolasefibronectin 1, Atype isoform 3 and (6671539,ankyrin repeat 39.79 domains kDa, 8.3) 1 3 2 (10%)(6%) 284154 fibronectin(13385328, type38.46 3 kDa,and an 7.0)kyrin repeat domains 1 3 (10%) 154 (13385328,acetyl-Coenzyme 38.46 AkDa, acetyltransferase 7.0) 2 (30348956, 2 (7%) 101 acetyl-Coenzyme41.73 kDa, 6.6) A acetyltransferase 2 (30348956, 2 (7%) 101 11 Vijay SB12- 41.73aldo-keto kDa, reductase 6.6) family 1, member B7 (6753148, 14 (55%) 3687 11 Vijay2D SB12-91-11 aldo-keto36.12 kDa, reductase 6.5) family 1, member B7 (6753148, 14 (55%) 3687 band2D 44 91-11 36.12cytochrome kDa, 6.5) c-1 (13385006, 35.53 kDa, 9.2) 8 (32%) 1868 band 44 cytochromevoltage-dependent c-1 (13385006, anion channel 35.53 2 kDa, (6755965, 9.2) 32.34 86 (32%)(24%) 1868 699 voltage-dependentkDa, 7.4) anion channel 2 (6755965, 32.34 6 (24%) 699 kDa,phosphoglycerate 7.4) kinase 2 (13654249, 45.25 kDa, 6.4) 5 (25%) 385 phosphoglycerateC-terminal fragment kinase 2 (13654249, 45.25 kDa, 6.4) 5 (25%) 385 C-terminalenoyl coenzyme fragment A hydratase 1, peroxisomal (7949037, 2 (12%) 328 enoyl36.44 coenzymekDa, 7.6) A hydratase 1, peroxisomal (7949037, 2 (12%) 328 12 Vijay SB12- 36.44cysteine-rich kDa, 7.6) secretory protein 1 (31981914, 28.52 kDa, 10 (34%) 2413 12 Vijay2D SB12-91-12 cysteine-rich6.4) secretory protein 1 (31981914, 28.52 kDa, 10 (34%) 2413 band2D 45 91-12 6.4)PREDICTED: alpha-enolase-like isoform 7 5 (20%) 735 band 45 PREDICTED:(309265176, 47.62 alpha-enolase-like kDa, 6.1) isoform 7 5 (20%) 735 (309265176,glutathione S-transferas 47.62 kDa,e, 6.1) mu 5 (6754086, 27.02 kDa, 3 (18%) 125 glutathione6.8) S-transferase, mu 5 (6754086, 27.02 kDa, 3 (18%) 125 13 Vijay SB12- 6.8)cysteine-rich secretory protein 1 (31981914, 28.52 kDa, 9 (34%) 1384 13 Vijay2D SB12-91-13 cysteine-rich6.4) secretory protein 1 (31981914, 28.52 kDa, 9 (34%) 1384 band2D 46 91-13 6.4)phosphatidylethanolamine binding protein 1 (84794552, 4 (40%) 414 band 46 phosphatidylethanolamine20.99 kDa, 5.2) binding protein 1 (84794552, 4 (40%) 414 20.99 kDa, 5.2) page 8 page 8

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coiled-coil domain containing 44 (39930435, 32.75 2 (17%) 284 kDa, 8.4) 14 Vijay SB12- ropporin, rhophilin associated protein 1 (13507710, 12 (75%) 4236 2D 91-14 24.16 kDa, 5.1) band 51 phosphatidylethanolamine binding protein 1 (84794552, 12 (80%) 3336 20.99 kDa, 5.2) peroxiredoxin 2 (31560539, 22.01 kDa, 5.2) 5 (24%) 1090 15 Vijay SB12- phosphoglycerate mutase 2 (9256624, 28.98 kDa, 8.7) 12 (40%) 2283 2D 91-15 dodecenoyl-Coenzyme A delta isomerase (3,2 trans- 3 (21%) 1542 band 60 enoyl-Coenyme A isomerase) (31981810, 32.52 kDa, 9.1) lysophospholipase-like 1 (22122621, 26.61 kDa, 7.8) 3 (15%) 417 enoyl Coenzyme A hydratase, short chain, 1, 3 (16%) 390 mitochondrial (29789289, 31.85 kDa, 8.8) hemoglobin alpha 1 chain (6680175, 15.13 kDa, 8.0) 3 (28%) 132 hypothetical protein LOC67507 (13385716, 27.26 kDa, 2 (13%) 121 8.6) electron transferring flavoprotein, beta polypeptide 2 (8%) 108 (38142460, 27.83 kDa, 8.2) adenylate kinase 2 isoform a (77020262, 26.74 kDa, 2 (9%) 94 6.3) outer dense fiber of sperm tails 1 (6679164, 29.86 kDa, 2 (12%) 84 8.3) 16 Vijay SB12- glutathione peroxidase 5 (6754062, 25.59 kDa, 8.6) 6 (31%) 212 2D 91-16 PARK2 co-regulated (66392180, 27.88 kDa, 8.9) 2 (8%) 163 band 63 progesterone receptor membrane component 2 (18%) 135 (31980806, 21.80 kDa, 4.4) thymidylate kinase isoform 1 (15778566, 24.19 kDa, 3 (14%) 83 5.7) 17 Vijay SB12- pyruvate kinase 3 (31981562, 58.38 kDa, 7.2) 12 (32%) 2253 2D 91-17 aldo-keto reductase family 1, member B7 (6753148, 11 (39%) 991 band 82 36.12 kDa, 6.5) 152 aldehyde dehydrogenase 4 family, member A1 2 (6%) 312 (34328415, 62.23 kDa, 8.6) aspartyl aminopeptidase (31560449, 52.74 kDa, 7.0) 4 (16%) 257 glyceraldehyde-3-phosphate dehydrogenase, 2 (8%) 226 spermatogenic (6679939, 48.10 kDa, 8.2) A-kinase anchor protein 4 isoform a (110347481, 95.56 4 (5%) 173 kDa, 6.6) tektin-5 (38080173, 64.12 kDa, 8.1) 3 (11%) 143 18 Vijay SB12- 3-oxoacid CoA transferase 2A (11968160, 56.95 kDa, 12 (29%) 2271 2D 91-18 9.1) band 83 glutamine synthetase (31982332, 42.83 kDa, 6.6) Report 1854 11 (33%) Vijay/Ramdas 1761 A-kinase anchor protein 4 isoform a (110347481, 95.56 11 (16%) 1248 kDa, 6.6) glucose phosphate isomerase 1 (6680067, 62.96 kDa, 8 (18%) 1113 7.8) page 9 tektin-5 (38080173, 64.12 kDa, 8.1) 8 (21%) 700 tubulin, alpha 8 (8394493, 50.70 kDa, 5.0) 3 (10%) 530 voltage-dependent anion channel 2 (6755965, 32.34 4 (17%) 291 kDa, 7.6) PREDICTED: similar to Protein C9orf138 (38086995, 4 (11%) 290 54.45 kDa, 8.5) dihydrolipoamide dehydrogenase (31982856, 54.75 3 (9%) 207 kDa, 8.0) interleukin 4 induced 1 (6753872, 70.38 kDa, 6.3) 3 (5%) 193 tubulin, beta, 2 (22165384, 50.26 kDa, 4.8) 2 (5%) 123 tektin 3 (62078759, 57.09 kDa, 6.6) 2 (5%) 115 19 Vijay SB12- Clusterin (7304967, 52.17 kDa, 5.3) 7 (13%) 1798 2D 91-19 thioredoxin-like 1 (31543902, 32.62 kDa, 4.8) 4 (15%) 413 band 86 eukaryotic translation elongation factor 1 delta isoform 2 (16%) 292 b (54287684, 31.39 kDa, 5.0) succinate-Coenzyme A ligase, ADP-forming, beta 2 (4%) 185 subunit (46849708, 50.42 kDa, 6.6) phosphoglycolate phosphatase (40254507, 34.98 kDa, 4 (17%) 178 5.2) 20 Vijay SB12- endoplasmic reticulum protein ERp29 precursor 7 (29%) 3019 2D 91-20 (19526463, 28.86 kDa, 5.9) band 87 hydroxyprostaglandin dehydrogenase 15 (NAD) 6 (25%) 1385 (6680263, 28.99 kDa, 5.7) cutC copper transporter homolog (13384952, 28.26 6 (32%) 537 kDa, 7.6) 21 Vijay SB12- glyceraldehyde-3-phosphate dehydrogenase, 17 (52%) 8891 2D 92-24 spermatogenic (6679939, 48.10 kDa, 8.2) band 30 ubiquinol cytochrome c reductase core protein 2 13 (41%) 2930 (22267442, 48.26 kDa, 9.3) acetyl-Coenzyme A acetyltransferase 1 precursor 5 (17%) 1158 (29126205, 45.13 kDa, 8.7) lactate dehydrogenase 3, C chain, sperm specific 9 (29%) 898 (7305229, 36.23 kDa, 8.4) glutathione peroxidase 4 isoform 1 precursor 9 (43%) 570 (90903233, 29.80 kDa, 10.3) acetyl-Coenzyme A acyltransferase 2 (29126205, 42.26 8 (37%) 559 kDa, 8.3) A-kinase anchor protein 4 isoform a (110347481, 95.56 4 (8%) 282 kDa, 6.6) Aminomethyltransferase (62000670, 44.44 kDa, 8.9) 2 (15%) 120

page 10 Report 1854 Vijay/Ramdas

glucose phosphate isomerase 1 (6680067, 62.96 kDa, 8 (18%) 1113 7.8) tektin-5 (38080173, 64.12 kDa, 8.1) 8 (21%) 700 tubulin, alpha 8 (8394493, 50.70 kDa, 5.0) 3 (10%) 530 voltage-dependent anion channel 2 (6755965, 32.34 4 (17%) 291 kDa, 7.6) PREDICTED: similar to Protein C9orf138 (38086995, 4 (11%) 290 54.45 kDa, 8.5) dihydrolipoamide dehydrogenase (31982856, 54.75 3 (9%) 207 kDa, 8.0) interleukin 4 induced 1 (6753872, 70.38 kDa, 6.3) 3 (5%) 193 tubulin, beta, 2 (22165384, 50.26 kDa, 4.8) 2 (5%) 123 tektin 3 (62078759, 57.09 kDa, 6.6) 2 (5%) 115 19 Vijay SB12- Clusterin (7304967, 52.17 kDa, 5.3) 7 (13%) 1798 2D 91-19 thioredoxin-like 1 (31543902, 32.62 kDa, 4.8) 4 (15%) 413 band 86 eukaryotic translation elongation factor 1 delta isoform 2 (16%) 292 b (54287684, 31.39 kDa, 5.0) succinate-Coenzyme A ligase, ADP-forming, beta 2 (4%) 185 subunit (46849708, 50.42 kDa, 6.6) phosphoglycolate phosphatase (40254507, 34.98 kDa, 4 (17%) 178 5.2) 20 Vijay SB12- endoplasmic reticulum protein ERp29 precursor 7 (29%) 3019 2D 91-20 (19526463, 28.86 kDa, 5.9) band 87 hydroxyprostaglandin dehydrogenase 15 (NAD) 6 (25%) 1385 (6680263, 28.99 kDa, 5.7) cutC copper transporter homolog (13384952, 28.26 6 (32%) 537 kDa, 7.6) 21 Vijay SB12- glyceraldehyde-3-phosphate dehydrogenase, 17 (52%)153 8891 2D 92-24 spermatogenic (6679939, 48.10 kDa, 8.2) band 30 ubiquinol cytochrome c reductase core protein 2 13 (41%) 2930 (22267442, 48.26 kDa, 9.3) acetyl-Coenzyme A acetyltransferase 1 precursor 5 (17%) 1158 (29126205, 45.13 kDa, 8.7) lactate dehydrogenase 3, C chain, sperm specific 9 (29%) 898 (7305229, 36.23 kDa, 8.4) glutathione peroxidase 4 isoform 1 precursor 9 (43%) 570 (90903233, 29.80 kDa, 10.3) acetyl-Coenzyme A acyltransferase 2 (29126205, 42.26 8 (37%) 559 kDa, 8.3) A-kinase anchor protein 4 isoform a (110347481, 95.56 4 (8%) 282 kDa, 6.6) Aminomethyltransferase (62000670, 44.44 kDa, 8.9) 2 (15%) 120

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