IMPORTANCE OF THE MAMMAL - SPECIFIC PROTEIN PHOSPHATASE ISOFORM,
PPP1CC2, IN SUPPORTING SPERMATOGENESIS AND SPERM FUNCTION
A thesis submitted
To Kent State University in partial
Fulfillment of the requirements for the
Degree of Master of Science
By
Sabyasachi Sen
December 2014
Thesis written by
Sabyasachi Sen
B.Tech. West Bengal University of Technology, 2008
M.Tech. West Bengal University of Technology, 2011
M.S., Kent State University 2014
Approved by
______Srinivasan Vijayaraghavan, Professor, Ph.D., Biological Sciences, Masters Advisor
______Laura G. Leff, Acting Chair, Ph.D., Department of Biological Sciences
______James L. Blank, Interim Dean, Ph.D., College of Arts and Sciences
TABLE OF CONTENTS
LIST OF FIGURES ……………………………………………………………………………...v
LIST OF TABLES ….…………………………………………………………...... ix
ACKNOWLEDGEMENTS………………………………………………………………………x
ABSTRACT ...... 1 CHAPTER I INTRODUCTION...... 2 1.1 Testis ...... 2
1.2 Spermatogenesis: ...... 3
1.2.1 Detailed Stages of Spermatogenesis: ...... 4
1.2.2 Regulation of spermatogenesis: ...... 6
1.3 Sperm motility: ...... 9
1.4 Protein Phosphatase and their role in sperm function: ...... 11
AIM ...... 15
CHAPTER II METHODS: ...... 16 Design of the PPP1CC2 transgene construct used for the making of the STZ-line for
analysis ...... 16
Genotyping and developing the STZ-line ...... 17
i
Preparation of mouse testis protein extracts:...... 21
Bradford Protein Assay ...... 22
Preparation of mouse sperm extracts: ...... 22
Western Blot Analysis ...... 22
Densitometry analysis: ...... 24
Protein Phosphatase assay: ...... 24
Isolation of RNA, RT-PCR, and Q-PCR: ...... 25
Sperm motility analysis ...... 26
Waveform Analysis ...... 27
Chapter III RESULTS ...... 28 1. Does overexpression of PP1CC1 or PP1CC2 affect spermatogenesis and sperm
function? ...... 28
Background and rationale: ...... 28
1.1 PPP1CC2 levels in testis of STZ (++/TgTg) and wild type (+/+) mice...... 29
1.2 PPP1CC2 protein expression in sperm of STZ-line (++/TgTg) and Wild type (+/+)
control...... 31
1.3 PPP1CC2 mRNA levels in testis of Wild type (+/+) and STZ (++/TgTg) animals. ... 34
1.4 Testis Weight, Sperm Number and Fertility of Wild type (+/+), STZ (++/TgTg
PPP1CC2) TW (++/Tg PPP1CC1) ...... 37
1.5 Analysis of sperm motility Wild type (+/+) and STZ-line (++/TgTg) ...... 39
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1.6 TW-line (++/Tg PPP1CC1) showed a reduction in PPP1CC2 protein levels in sperm in
comparison to STZ-line (++/TgTg PPP1CC2), and Wild type control...... 42
1.7 Protein phosphatase 1 (PP1) activity of Wild type (+/+), STZ (++/TgTg PPP1CC2),
and TW (++/Tg PPP1CC1)...... 45
1.8 Protein Kinase A (PKA) mediated serine-threonine phosphorylation levels of substrates
in sperm of Wild type (+/+), Heterozygous (+/-), STZ (++/TgTg PPP1CC2), and TW (++/Tg
PPP1CC1)...... 50
1.9.1 Spz-1 protein expression in testis of STZ-line (++/TgTg) and Wild type (+/+) control.
...... 52
1.9.3 PPP1R11 (I3) protein expression in testis of STZ-line (++/TgTg) and Wild type (+/+)
control...... 55
SUMMARY ...... 57
CHAPTER IV DISCUSSION: ...... 58 A. Optimal levels and maintenance of PPP1CC2 protein is important for maintaining
normal spermatogenesis, sperm function and fertility rates...... 58
B. High levels of regulation of PP1 activity is of utmost importance for maintaining normal
sperm function and fertility rates ...... 59
C. Lack of Protein Kinase A (PKA) mediated serine-threonine phosphorylation might be the
major cause for the TW (++/Tg PPP1CC1) line being sub-fertile...... 60
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D. Increased expression of Spermatogenic Zip protein -1 (Spz-1) might lead to regulation of
PPP1CC2 ...... 60
E. Protein phosphatase regulatory subunit 11 (PPP1R11) might also regulate PPP1CC2 .... 61
BIBLIOGRAPHY ...... 62
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LIST OF FIGURES
CHAPTER I
Figure 1. A cross section of the mammalian testis………………………………………………..4
Figure 2. Schematic representation of various stages of spermatogenesis………………………..7
Figure 3. Schematic representation of different forms of alternate Splicing…………………….10
Figure 4. Schematic representation of the difference between progressive and hyperactive motility in sperms………………………………………………………………………………..12
Figure 5: Schematic representation of PPP1 and its four main isoforms. PPP1/PP1 shows high homology in the catalytic core with PP2A and PP2B……………………………………………13
Figure 6. Alternative spicing of PPP1CC gene producing PPP1CC2 and PPP1C1 transcripts….15
CHAPTER II
Figure 7. The transgene construct driven by testis specific human Pgk2 (Phospho glycerate kinase -2) promoter. Ppp1cc2 sequence used lacked the 5’ and 3’ UTRs. The SV40 PolyA signal sequence was added 3’ of the cDNA construct………………………………………………….18
Figure 8. Mating scheme for obtaining a single copy of the PPP1 CC2 transgene, on a wild type background……………………………………………………………………………………….19
Figure 9. Test cross mating scheme to identify the genotype of the male parent………………..20
Figure 10. Mating scheme to obtain two copies of the PPP1CC2 transgene, on a wild type background (STZ – line)…………………………………………………………………………21
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Figure 11. Test cross mating scheme to identify the genotype of the male parent………………22
Chapter III
Figure 12A.Western blot analysis of PPP1CC2 levels in testis of Wild type (+/+) and STZ
(++/TgTg)………………………………………………………………………………………..33
Figure 12B. The same blot was re-probed with β-tubulin for equal loading control……………33
Figure 13A.Western blot analysis of PPP1CC2 levels in sperm of Wild type (+/+) control and
STZ (++/TgTg)…………………………………………………………………………………..35
Figure 13B. The same blot was re-probed with β-tubulin for equal loading control……………35
Figure 13C. Relative PPP1CC2 protein levels (in terms of percentage) in testis of wild type
(+/+) and STZ (++/TgTg)………………………………………………………………………..36
Figure 14 A. Schematic representation of design of primers used to check for total PPP1CC2 mRNA levels……………………………………………………………………………………..37
Figure 14 B. Schematic representation of design of primers used to check for endogenous
PPP1CC2 mRNA levels………………………………………………………………………….39
Figure 14C. Relative PPP1CC2 mRNA levels in testis (In terms of percentage) of Wild type
(+/+) and STZ (++/TgTg)………………………………………………………………………..39
Figure 14D: Relative endogenous PPP1CC2 mRNA levels in testis (in terms of percentage) of
Wild type (+/+) and STZ (++/TgTg)...... 40
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Figure 15A. Total Percentage motility and Progressive motility of sperms in Wild type (+/+) and
STZ (++/TgTg)…………………………………………………………………………………..42
Figure 15B. Flagellar beat waveform analysis of Heterozygous (+/-), STZ (++/TgTg PPP1CC2) and TW (++/Tg PPP1CC1)………………………………………………………………………43
Figure 16 A. Western blot analysis of PPP1CC2 protein expression in sperm of Wild type (+/+),
TW (++/PPP1CC1Tg) and STZ (++/PPP1CC2TgTg)…………………………………………..46
Figure 16 B. The same blot was re-probed with β-tubulin for equal loading control…………...46
Figure 16 C. Showing presence of PPP1CC1 protein in sperm in TW-line (++/Tg PPP1CC1) and
T-line (--/Tg PPP1CC1), and lack of it in Wild type (+/+) and STZ-line (++/TgTg PPP1CC2) sperm, as expected, when probed with anti-PPP1CC1 antibody………………………………...46
Figure 16D. Relative PPP1CC2 protein levels (in terms of percentage) in sperm of Wild type
(+/+), STZ (++/TgTg) and TW (++/Tg PPP1CC1)……………………………………………...47
Figure 17A. Total protein phosphatase activity in the soluble cytosolic sperm fraction
(Supernatant) Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms……………………49
Figure 17B. Total protein phosphatase activity in the insoluble cytosolic sperm fraction (Pellet) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms……………………………...…51
Figure 17C. Total protein phosphatase 1 activity in the soluble cytosolic sperm fraction
(Supernatant) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms…………………52
Figure 17D. Total protein phosphatase 1 activity in the insoluble cytosolic sperm fraction
(Pellet) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms………………………..53
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Figure 18A. Western blot analysis of profile of substrates phosphorylated by Protein Kinase A
(PKA) on their serine-threonine residues of Heterozygous (+/-), Wild Type (+/+), TW
(++/TgPPP1CC1) and STZ (++/TgTg PPP1CC2)……………………………………………….54
Figure 18B. Same blot was re-probed with loading control Tubulin to show equal amount of sperm were loaded in each lane………………………………………………………………….55
Figure 19A.Western blot analysis of Spz-1 levels in testis of Wild type (+/+) and STZ
(++/TgTg)………………………………………………………………………………………..56
Figure 19B. Same blot was re probed with anti-PPP1CC2 antibody to check levels of PPP1CC2 expression, which were found to be equal……………………………………………………….56
Figure 19C. Same blot was re probed with β-tubulin antibody for loading control……………..56
Figure 19D. Relative Spz-1 protein levels (in terms of percentage) in testis of Wild type (+/+) and STZ (++/TgTg)……………………………………………………………………………...57
Figure 20A. Western blot analysis of PPP1R11 levels in testis of Wild type (+/+) and STZ
(++/TgTg)………………………………………………………………………………………..58
Figure 20B. Relative PPP1R11 protein levels (in terms of percentage) in testis of Wild type (+/+) and STZ (++/TgTg)……………………………………………………………………………...60
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LIST OF TABLE
CHAPTER I
Table 1. Reference table for genotype symbol…………………………………………………..16
CHAPTER II
Table 2. List of primers used to check the genetic background of animals……………………...23
Table 3. List of primary antibodies used for detection…………………………………………..26
Table 4. List of primers used for QPCR…………………………………………………………28
Chapter III
Table 5. Comparison of Testis Weight and Sperm Number between Wild type (+/+), STZ
(++/TgTg) and TW (++/Tg)……………………………………………………………………...40
Table 6: Fertility data of Wild type (+/+) and STZ (++/TgTg)………………………………….41
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Acknowledgement:
I would like to thank my advisor Dr. Srinivasan Vijayaraghavan for providing me the opportunity to further my knowledge and be part of a supportive and exciting lab. Under his training I have been able to achieve a firm foothold in field of research.
I would also like to thank my committee members Dr. Wen-Hai Chou and Dr. Douglas Kline for their wisdom, expert and insight during my time as a graduate student. I would specially like to thank Dr. Wen-Hai Chou for providing me with all his expertise in writing the review.
I would also like to thank my lab mates Tejasvi Dudiki, Nidaa Joudeh Awaja, Rahul Bhattacharya,
Suranjana Goswami and Shawn Davis for their overall support. I would specially like to thank
Tejasvi Dudiki and Santanu De for all their help and guidance. I am thankful to Donna Warner for being there always.
Without support from my family I could not have gone this far in my education. So with much gratitude, I would like to thank my parents and my sister, for providing me with all the help emotionally and financially.
Finally, thank you Purva Gawde for always cheering me up when the pressures of graduate school got me down. I am grateful to have her encouragement and humor every day.
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ABSTRACT:
PPP1CC1 and PPP1CC2 are alternatively spliced transcripts of PPP1CC gene. They are mostly identical, with difference only at their extreme C-termini. PPP1CC1 is ubiquitously expressed in somatic cells, while PPP1CC2 is germ cell specific. Complete deletion of PPP1CC leads to sterility due to impaired sperm morphogenesis. It has been previously documented, that fertility can be restored by optimum expression of PPP1CC2, by incorporating PPP1CC2 transgene on a PPP1CC null (-/-) background. It has also been seen that higher activity levels of PP1, renders sperm immotile but low activity levels initiate motility, as seen in caudal sperms (23). So, we wanted to determine if overexpressing PPP1CC2 in wild type (+/+) testis affected spermatogenesis and sperm function. Mouse line was generated, having two copies of PPP1CC2 transgene on a wild type background, and was compared to wild type controls. It was found that PPP1CC2 protein as well as activity levels were highly regulated both in testis and sperm, and remained unchanged and comparable to that of wild type. Sperm were phenotypically indistinguishable from their wild type controls in terms of sperm morphology, motility and number. PPP1CC2 mRNA levels however showed a doubling in their levels in comparison to the wild type controls. Meanwhile, PPP1CC2 protein levels were found out to be drastically reduced in the mouse line, having one copy of
PPP1CC1 transgene on a wild type background. There was no evidence of Protein Kinase-A mediated serine-threonine phosphorylation in these mice. Mice having one copy of PPP1CC1 transgene on a wild type background was found to be sub-fertile. Our studies suggest, perhaps, the high level of regulation of PPP1CC2 and maintenance of optimal levels of PPP1CC2 expression is crucial for maintaining normal spermatogenesis and sperm function.
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CHAPTER I
INTRODUCTION
1.1 Testis Starting from a self-renewing stem cell pool, male germ cells develop in the seminiferous tubules of the testes from puberty throughout life until old age. The mammalian male reproductive system comprises of the testis (Figure 1), encased in Tunica albuginea formed of connective tissue and interconnected ducts. Testes, besides containing germ cells, also contain somatic cells like Sertoli cells, interstitial cells, Leydig cells, mast cells, macrophages and peritubular myoid cells. The function of testis is both endocrine and exocrine in nature, by means of secreting male sex hormones and formation of sperm by the process of spermatogenesis. Seminiferous tubules, converges onto the rete testis, and form a system of collective ducts called the epididymis, sperm develop in the environment of the seminiferous tubule (3). In the seminiferous tubule, the spermatogonal cells and post-meiotic germ cells are present in different regions, with, the spermatogonal cells located in the basal compartment, while, the post-meiotic germ cells are located in the adluminal compartment. The post-meiotic germ cells stay in close association with the Sertoli cells in the adluminal compartment (2, 6). They do not receive direct nutrition from or come in contact with the blood stream of the basal compartment, thus, making them completely dependent on Sertoli cells for their existence (4, 7). Adjoining Sertoli cells also prevent large molecules such as antibodies from entering the lumen of seminferous epithelium, by means of the blood testis-barrier. The peritubular myoid cells provide structural support to the seminiferous tubules.
2
Figure 1. A cross section of the mammalian testis (30)
1.2 Spermatogenesis: Male germ cell differentiation is a highly regulated, complex process that takes place within the seminiferous tubules. The complete process of sperm development is called spermatogenesis. The generation of spermatozoa capable of fertilizing an egg involves various complex processes starting in the male gonads and reaching completion in the female reproductive tract (1). The entire process of maturation of spermatozoa, takes place in the seminiferous tubule. Spermatogenesis can be divided into three phases. (i) spermatogonial proliferation, (ii) meiosis of spermatocytes and
(iii) spermiogenesis, a morphological process converting haploid spermatids to spermatozoa
3
followed by spermiation, release of mature testicular sperm in the lumen in testis(2). Overall, spermatogenesis is highly regulated by complex signaling mechanisms involving gene expression patterns leading to the continuous production of spermatozoa starting from puberty through the reproductive life span of the male (2).
During spermatogenesis male germ cells undergo complex differentiation where morphological alterations lead to the formation of differentiated sperm. Spermatogonial stem cell (SSC) located next to the germinal epithelium undergo mitosis to give rise to two daughter cells. Out of the two, one daughter cell enters spermatogenesis and the other remains as a SSC, to undergo self-renewal.
The daughter cell next undergoes spermatogenesis and further rounds of mitosis – six in rats (2) and two or more in humans (2,5) before undergoing meiosis.
1.2.1 Detailed Stages of Spermatogenesis: The process of spermatogenesis starts with A1 spermatogonia located adjacent to the outer basement of the seminiferous epithelium. This A1 spermatogonia divide mitotically to generate two types of cells- A1 spermatogonia, which are essentially stem cells that keeps regenerating themselves, and a paler A2 spermatogonia. The A2 spermatogonia divide to produce A3 spermatogonia, which then beget type A4 spermatogonia, which beget intermediate spermatogonia. This intermediate spermatogonia further divides mitotically to produce type B spermatogonia. This type B spermatogonia then divide to generate the primary spermatocyte, which enter the first meiotic division of spermatogenesis (2). The process of spermatogenesis starts with A1 spermatogonia located adjacent to the outer basement of the seminiferous epithelium. This
A1 spermatogonia divide mitotically to generate two types of cells- A1 spermatogonia, which are
4
essentially stem cells that keeps regenerating themselves, and a paler A2 spermatogonia. The A2 spermatogonia divide to produce A3 spermatogonia, which then beget type A4 spermatogonia, which beget intermediate spermatogonia. This intermediate spermatogonia further divides mitotically to produce type B spermatogonia. This type B spermatogonia then divide to generate the primary spermatocyte, which enter the first meiotic division of spermatogenesis (3). As division of cells progress from type A spermatogonia to spermatid stage, the cells start moving gradually from the basement membrane towards the lumen of the seminiferous tubule. Thus, each type of cells can be found at distinct layers of the tubule. Following, meiotic and mitotic division, cells fail to complete cytokinesis, and form cytoplasmic bridges. These cytoplasmic bridges are means by which the cells remain connected to each other. Hundreds or even thousands of cells remain connected to each other at any distinct layer of the tubule by means of these cytoplasmic bridges. Primary (preleptotene) spermatocytes, generates pachytene and diplotene spermatocytes, as it enters meiosis, followed by formation of haploid step 1 spermatids. Spermatids are essentially, round unflagellated cells, which undergo the process of morphological differentiation, known as spermiogenesis. Spermiogenesis results in the formation of a complete structurally mature but functionally immature spermatozoa. The major characteristics of spermiogenesis is the formation of acrosomal cap over the nucleus; generation of flagellum form the centriole located opposite to the nucleus; condensation of nucleus; ejection of cytoplasmic droplet; and finally the formation of mitochondria as a ring around the flagellum. After the completion of spermiogenesis, structurally mature spermatozoa are released into the lumen of the seminiferous tubule, by a process, known as spermiation. It involves the retraction of the cytoplasm of the sertoli cells around the spermatid head; removal of excess cytoplasm of the spermatid, thus, resulting into a streamlined
5
spermatozoan, spermatid extension towards the lumen of the seminiferous tubule, and finally, disengagement of the sperm from the sertoli cell into the lumen of the tubule(3).
Figure 2: Schematic representation of various stages of spermatogenesis.
1.2.2 Regulation of spermatogenesis: Spermatogenesis reaches its conclusion with the formation a morphologically and functionally unique cell type, the spermatozoa. Spermatogenesis involves cell to cell contact, cross‐talk and elaborate signaling between them, achieved by means tight control and regulation of gene
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expression. This regulation is achieved in three possible ways: intrinsic, interactive and extrinsic form (8).
The Intrinsic form regulates expression of genes and protein, which play important role in the process of germ cell development and differentiation. The interactive form plays the role of integrating external information (hormonal) and modulates and relay the information in between the germinal epithelium and between the germ cells at various stages of development, at the level of the Sertoli cell. Finally, the extrinsic level involving the hormonal or endocrine regulation of gene expression and signaling mechanism in somatic cells of seminiferous tubules which then modulates the activity of the germ cells.
Intrinsic regulation of gene expression during spermatogenesis can be regulated at the following levels: Transcriptional, Posttranscriptional and Translational control level. Up‐regulation of general transcription factors as well as testis specific factors for developmental expression of gene leads to high levels of transcription in the testis. General transcription factors like TFIID, TFIIB,
TBP and RNA Polymerase II are overexpressed in testis underscoring the need for transcription.
Among the numerous testis specific factors predicted, the most important is CREM. CREM is a c‐
AMP responsive element binding protein that binds to CRE (c‐AMP response elements) on promoter of target genes. Some of the know target of CREM are transition protein‐I (9), ACE (10);
CYP51. Several CREM isoforms formed due to alternative splicing are found in the testis, but the activator form is the most prevalent form. They associate with testis specific activators ACT and
KIF17b (11) causing transcriptional activation of gene expression in testis (12). Other known testis specific factors are Spz1 (13) and A‐Myb (14). Thus, expression of testis-specific genes results due to the recruitment of testis specific elements in the promoter or by utilization the combination of unique transcription factor that acts through pre‐existing promoter elements.
7
Two most prevalent methods of posttranscriptional modification in testis are: Alternative splicing: Alternative splicing process leads to genome diversification, thus, enhancing the coding capacity of the genome and in turn presenting a new level of gene expression. Alternative splicing in testis occurs through the following or a combination of methods - Alternate exon skipping or inclusion, giving rise to new, variable functional domains to serve different specific roles in testis. Use of alternate promoters, leading to change at the N-terminal of protein, due to the presence of an alternative transcription start site or inclusion (or exclusion) of novel exons (at
5’ end).Use of alternate Poly A adenylation sites (within a intron): Resulting in exon skipping and thus, effecting the 3’ end of the transcript or C‐tail of the protein (Figure 3). One or a combination of the above mentioned factors can result in testis specific variant or protein expression (15).
Figure 3: Schematic representation of different forms of alternate Splicing (adapted from
Reference 15 and from the thesis of Shandilya Rmadass “ROLE OF PP1γ2 BINDING
PARTNERS IN SPERMATOGENESIS AND SPERM FUNCTION.” December 2012)
8
Representation of eight common types of alternative transcript events each capable of producing multiple mRNA isoforms through alternative splicing, alternative cleavage and polyadenylation and/or alternative promoter usage.
Regulation of spatial and temporal expression of genes in testis during spermatogenesis and germ cell development, leads to the uncoupling of translation from transcription. Transcription ceases mid‐spermiogenesis in round spermatids. Thus proteins required during late spermatogenesis are expressed during earlier stages and stored as translationally repressed messenger ribonucleoprotein particles (mRNPs). These mRNAs are delayed in their translational and only coded when the specific product are required during late spermiogenesis (16).
1.3 Sperm motility: In mammals sperm motility is necessary the primary prerequisite, for any fertilization to occur.
The seminiferous epithelium can be divided into three broad regions - caput (head), corpus (body) and cauda (tail). Sperm in the caput region stay immotile and gain the capacity for motility while their transit through the corpus to the caudal of vasa differentia, where they stay in a calm state due to the viscosity drag of the caudal fluid. Sperm acquire motility, once, they are deposited into the female reproductive tract, and they move through the female tract and penetrate cumulus oophorous and zona pellucida (2).
There are two kinds of motility in sperm. Progressive motility is characterized by vigorous and rapid forward movement, accompanied with symmetrical tail beat, while immature spermatozoa exhibit erratic beats without any net propulsion. Sperm progressive motility is established during its transit through epididymis. Capacitation is the final stage of sperm maturation that determines
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the ability of the sperm to fertilize eggs. Sperm undergo biochemical and morphological changes, such as efflux of cholesterol, increased levels of cAMP due to increased activity of adenylyl cyclase, an increase in protein tyrosine phosphorylation levels, increase in intracellular Ca2+ and pH and changes in the biochemical properties of the sperm plasma membrane (17). Due to all these changes, sperm acquire hyperactivated motility, and are able to bind to the zona pellucida. High amplitude asymmetrical beats of tail; lateral vigorous movement of tail and circular or erratic trajectory of path are some of the characteristics of hyperactive motility (Figure 4) (2).
Figure 4 Schematic representation of the difference between progressive and hyperactive motility in sperms (adapted from reference 4) Progressive motility is characterized with vigorous and rapid forward movement, accompanied with symmetrical tail beat. High amplitude asymmetrical beats of tail; lateral vigorous movement of tail and circular or erratic trajectory of path are some of the characteristics of hyperactive motility.
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1.4 Protein Phosphatase and their role in sperm function: Protein phosphatases can be broadly classified into two kinds: they are phosphoprotein serine- threonine phosphatases and phosphoprotein tyrosine phosphatases. Phosphoprotein phosphatase 1
(PPP1) belongs to the class of serine-threonine phosphatases and are mammalian specific. Protein phosphatase 1 (PPP1) shares the same 280 amino acid catalytic core with Protein phosphatase 2A
(PP2A), and protein phosphatase 2B (calcineurin), while differing only at their N-and C-termini
(Figure 5) (18).
Figure 5: Schematic representation of PPP1 and its four main isoforms. PPP1/PP1 shows high homology in the catalytic core with PP2A and PP2B (reference 24) (Adapted from the thesis of Nilam Sinha- “Mammal specific protein phosphatase isoform,PPP1CC2, is essential for sperm function and male fertility”).
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Phospho protein Phosphatase 1 is highly conserved from yeast to humans. PPP1 has four isoforms, and the catalytic cores of all the four phosphatase isoforms are highly conserved. PPP1CC1 and
PPP1CC1 are spliced from the same gene involving utilization of Exon 7. PPP1CC1 retains the whole of Exon 7, while, PPP1CC2 retains only a portion of Exon 7 which joins Exon 8 (Figure 6).
They differ only at their C-terminus, with PPP1CC2 having a unique 21 amino acid tail. PPP1CC2 is the only unique testis specific isoform of PPP1 (Figure 6), while, PPP1α, PPP1β and PPP1CC1 are ubiquitously expressed. Previous research in has shown, PPP1CC2 to be the predominant isoform to be expressed in sperms, both in terms of high protein expression and enzyme activity.
It was also seen that their activity was higher in the sperms extruded from caput in comparison to the caudal sperm, thus, suggesting their role in epididymal maturation of sperm. To find out the role of PPP1CC2, the PP1CC gene was knocked out, which made male mice infertile, Testis showed spermatogenic defects and high degree of apoptosis (19).
PPP1CC gene knockout lacked both PPP1CC2 and PPP1CC1 isoforms In order to elucidate the isoform specific importance of the two isoforms. PPP1CC2 transgene driven by testis-specific promoter Phospho Glycerate-Kinase 2 was incorporated into mice lacking the PPP1CC gene (20).
Transgenic expression of PPP1CC2 in knockout mice was able to restore fertility and spermatogenesis (21). Research, in our lab (work done by my colleague Tejasvi Dudiki)(22) has also shown that similar expression of PPP1CC1 as a transgene in mice lacking PPP1CC gene, restores spermatogenesis and but sperm have reduced motility and males sub-fertile (22). These studies showed that that PPP1CC2 is indispensable for normal fertility and spermatogenesis.
Previous research has also showed that PP1 activity affects sperm motility. High catalytic activity renders them immotile but low activity levels initiate motility, as seen in caudal sperms (23).
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Figure 6. Alternative spicing of PPP1CC gene producing PPP1CC2 and PPP1C1 transcripts. (A) PPP1CC1 and PPP1CC2 are mammalian specific alternate spliced product of
PPP1CC gene, involving utilization of exon 7. While PPP1CC1 retains the whole of Exon 7,
PPP1CC2 retain only a part of it (internal splice site, GT) which joins Exon 8 due to the splicing event. (B) This alternate splice event generates a unique 21 amino acid tail exclusive to
PPP1CC2. (Adapted from the thesis of Nilam Sinha- “Mammal specific protein phosphatase isoform, PPP1CC2, is essential for sperm function and male fertility”).
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Table 1:
For all my data shown in the results, please refer to the table (Table 1) below for genotype symbols.
Reference table for genotype symbols
Symbols Used Meaning of the symbols
WT (++) Wild Type: Used as control, animals
having two copies endogenous PPP1CC2
gene.
STZ (++/TgTg) or (++/Tg+Tg+) or (++/TgTg Animal line with two copies of PPP1CC2
PPP1CC2) transgene in wild type mice.
Ht (+/-) or (+/- PPP1CC) Heterozygous/mice with one copy of
endogenous PPP1CC allele.
KO (-/-) or (-/- PPP1CC) Knockout for PPP1CC.
TW (++/Tg) or (++/Tg PPP1CC1) Mice with one copy of PPP1CC1
transgene incorporated on a wild type
background.
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AIM
Does overexpression of PPP1CC2 or PPP1CC1 affect spermatogenesis and sperm function?
- The approach was the generation and analysis of mice carrying four copies of PPP1CC2 –
two wild type endogenous alleles and two transgene copies of PPP1CC2 and mice
expressing PPP1CC1 as a transgene in a Ppp1cc wild type background.
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CHAPTER II
METHODS:
Design of the PPP1CC2 transgene construct used for the making of the STZ-line for analysis
Figure 7. The transgene construct driven by testis specific human Pgk2 (Phospho glycerate kinase -2) promoter. Ppp1cc2 sequence used lacked the 5’ and 3’ UTRs. The SV40 PolyA signal sequence was added 3’ of the cDNA construct (24).
Design of the construct
The transgene construct is driven by the human PGK-2 (Phospho glycerate kinase -2) promoter.
The PPP1CC2 coding region was followed by the SV 40 polydenylation sequence. The transgene construct was injected into the pronucleus of a fertilized mouse egg. The zygote was allowed to to form an embryo. The developed embryo was then implanted in the uterus of a pseudo pregnant foster female mouse. The offspring carrying transgene were crossed with PPP1CC2 containing wild type males or females to produce the STZ line for analysis.
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Genotyping and developing the STZ-line The first step was to establish the line, with one copy of PPP1CC2 transgene on a wild type background. A wild type male was crossed with a female with one copy of PPP1CC2 transgene on a heterozygous Ppp1cc (+/-) background. This mating resulted in mice with four different genetic background were obtained, they were, a wild type, a heterozygous, one copy of PPP1CC2 transgene on a heterozygous background and finally, the desired one copy of PPP1CC2 transgene on wild type background (Figure 8).
Figure 8. Mating scheme for obtaining a single copy of the PPP1 CC2 transgene, on a wild type background.
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Test cross to determine that PPP1CC2 transgene was hemizygous on a wild type background
Next, in order to confirm the presence of one copy of PPP1CC2 transgene on a wild type background, test crosses were set up, with wild type females. Test crosses, showing 50% of the total litter size to be positive for the PPP1CC2 transgene, suggesting that the parent contains one copy of the PPP1CC2 transgene, as seen in the mating scheme shown below (Figure 9).
Figure 9. Test cross mating scheme to identify the genotype of the male parent
Then males having one copy of PPP1CC2 transgene on wild type background were crossed with females with the same genetic background. From the breeding, animals with possibly three different genetic background were obtained, a wild type, one copy of PPP1CC2 transgene on wild type background, and lastly, a 25% probability of our desired line with two copies of PPP1CC2 on a wild type background (STZ – line)(Figure 10).
18
Figure 10. Mating scheme to obtain two copies of the PPP1CC2 transgene, on a wild type background (STZ - line)
Test cross to check homizygosity of the PPP1CC2 transgene on a wild type background
In order to check, for the homozygosity of the PPP1CC2, the animals obtained from the previous breeding cross, was subjected to test cross, with wild female. Animals homozygous for the
PPP1CC2 transgene on a wild type background when test crossed with a wild type female, showed a 100% presence of PPP1CC2 transgene, as expected (Figure 11).
19
Figure 11. Test cross mating scheme to identify the genotype of the male parent
Genotyping and Polymerase Chain Reaction:
For checking the genetic makeup of the mice, each ear punch samples were immersed in Lysis
Buffer (10 N NaOH, 0.5 N EDTA and distilled water) to extract DNA, 40mM Tris HCL was added to each sample after one hour. This was followed by polymerase chain reaction (PCR) to identify the genotype of each animal.
20
Table 2. List of primers used to check the genetic background of animals:
Primers Used Sequence
Intron IV Forward Primer 5’ CTC AGG CCA ATG CTG TCT 3’
Intron VI Reverse Primer 5’ ACT CAT AGC CAT CTT CAA CCA
3’
Exon 6 Forward Primer 5’ GTG GTT GAA GAT GGC TAT GA 3’
SV 40 Reverse Primer 5’ AAG CTG CAA TAA ACA AGT TGG
3’
Primers Exon 6 Forward and SV40 Reverse were used to detect the presence of the PPP1CC2 transgene, primers Intron IV and Intron VI were used to differentiate wild type from animals having one endogenous allele of PPP1CC gene.
Preparation of mouse testis protein extracts: Testis tissues were isolated from 6 week or mice. Isolated tissue were next homogenized in HB+ buffer (10 mM Tris‐HCl, pH 7.0, 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine‐HCl, 1 mM
PMSF, 0.1 mM N‐tosyl‐L‐phenylalanine chloromethyl ketone [TPCK], 0.1% [V/V] β mercaptoethanol). The homogenates were then subjected to centrifugation at 16,000×g for 20 min to remove insoluble material. After protein estimation (as mentioned below), 6x Laemmli sample buffer was added to the supernatants to a final concentration of 1X, then boiled for 5 min, and stored at ‐20ºC until use.
21
Bradford Protein Assay Protein estimation and serial dilution was performed with the supernatant testis extracts. The extracts were left for overnight precipitation with 20%TCA solution. They were then centrifuged at 12000 x g for 15 min at 40 0C and the protein pellets were air dried to be dissolved in 0.1 N
NaOH. The BSA standard was serially diluted with 0.1 N NaOH. The estimated protein values were used to plot an absorbance graph, and the linear absorbance equation was used to calculate the concentration of protein in each testis extract sample. These values were used for calculating the amount in volume of the extracts to be used for running the western blot gels.
Preparation of mouse sperm extracts: Caudal epididymal sperm were extruded and collected in 1X phosphate buffered (1x PBS) saline.
For counting, sperm were diluted (1:10) in purified water and 10 μl of the diluted sperm were loaded onto a Neubaumer Haematocytometer for sperm count, the whole sperm extracts were then subjected to centrifugation at 710 x g for 10 minutes. After discarding the supernatant (1X PBS), sperm pellet was re-suspended in 1% sodiumdodecylsulphate (SDS) in water. The dispersed pellets were sonicated on ice with three 15 second bursts at 20% amplitude (Misonix incorporated). Next, the resulting sperm sonicates were boiled for 5 min followed by centrifugation at 12,000xg for 15 min. The supernatant was collected and boiled with lamelli buffer ready to be loaded for western blot analysis.
Western Blot Analysis Testis and sperm protein extracts made in Laemmli sample buffer, were separated by 12% SDS‐
PAGE, and transferred to Immobilon‐P PVDF membranes (Millipore Corp, Billerica, MA, USA).
After blocking non‐specific binding sites with 5% nonfat dry milk in Tris‐buffered saline (TBS:
22
25 mM Tris‐HCl, pH 7.4, 150 mM NaCl), blots were incubated with primary antibodies (1:5000 dilution) overnight at 4°C. After washing, blots were incubated with anti‐rabbit/mouse secondary antibody (1 :5000 dilution) conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ,
USA) for 1 hr at room temperature. Blots were washed with TTBS twice for 15 min each and four times for 5 min each, and then developed with homemade ECL chemiluminescence preparation.
Table 3. List of primary antibodies used for detection:
Antibod Manufacturer Dilutio Secondar Detects y Used n y
antibody
Anti- Custom Made[Peptide sequence – (1:5000 Anti- PPP1CC2
PPP1CC VGSGLNPSIQKASNYRNNTVLYE]( ) rabbit
2 28)
Anti- Custom Made[Peptide sequence- TPPR- (1:4000 Anti- PPP1CC2
PPP1CC --GMIT—KQAKK----](28) ) rabbit
1
Anti - Epidomics (1:5000 Anti- Tubulin
αTubulin ) rabbit
Anti- Custom made[ Peptide sequence (1:1000 Anti- PPP1R11
PPP1R1 NAETGAGISETVTET](29) ) rabbit
1
Anti- Custom Made (1:50) Anti- Spz-1
Spz-1 rabbit
23
Anti- Cell Signalling (1:5000 Anti- Serine-
Phospho- ) rabbit threonine
Protein phosphorylatio
Kinase A n on substrates
by PKA
Densitometry analysis: To quantify and compare intensities of protein bands in western blots, Multi Gauge‐ver3.X (from
Fujifilm Inc.) image analysis software was used for densitometry. Bands that appeared to be of equal intensities by visual inspection were selected for densitometry analysis.
Protein Phosphatase assay: The buffer assay contained 2mM EDTA, 20mg/ml BSA, 400mM imidazole-HCL (pH 7.63), 2%
(v/v) β-mercaptoethanol. Two µl of sperm extract (2 X 105 sperm), 20µl of 1X Assay buffer, 2µl of 10mM MnCl2. Okadaic acid, a known selective inhibitor of PPP2A at a concentration 2nM (31) was added to check total PP1 activity. Next, the mixture was incubated at 30o C for 5 minutes.
After incubation was done, 10 µl of Phosphorylase a ( 32P) was added, and again incubated at 30o
C for 10 minutes. Then, all tubes containing the mixture were put on ice and finally, each reaction was stopped by adding 20% TCA and was kept on ice for 3-5 minutes. Next, all the mixtures were centrifuged at 12,000 X g at 4oC for 5 minutes. Lastly, 190 µl of the supernatant after the spinning down, was taken for each sample and added to 1ml dH2O in scintillation vials, and finally, were taken to the scintillation counter and readings were taken.
24
Isolation of RNA, RT-PCR, and Q-PCR: Total RNA from testes of adult mice (6 weeks or older) was isolated using TRI reagent (Sigma
Aldrich). Testes were homogenized in TRI reagent. Chloroform was added and then the homogenates were held on ice for 15 minutes followed by centrifugation at 12,000 g at 4o C. The top layer of the supernatant containing total RNA was collected. Then, isopropanol was added and the mixture was incubated at room temperature for 5 minutes, followed by centrifugation at 10,000
X g for 10 minutes at 4oC. After centrifuging, the pellet was saved and to it 75% ethanol was added and gently vortexed. The mixture was centrifuged at 7500 X g for 7 minutes at 4o C. The pellet was retained and semi dried, and was finally resuspended in RNAse free water, and concentrations determined. The estimated total RNA values were used to plot an absorbance graph, and the linear absorbance equation was used to calculate the concentration of RNA in each testis extract sample.
The samples were then diluted to a concentration of 200 ng/µl. The manufacturer’s protocol
(Qiagen) was followed for RT-PCR using 800 ng of template RNA. Two step RT-PCR was performed. In the first step, g DNA wipeout buffer, RNAse free water was added to remove any
DNA contamination, while in the second step, Reverse Transcriptase, RT buffer and Primer mix was added. Glyceraldehyde 3-phopshate dehydrogenase (GAPDH) was used as the control, for presence of the concentration of cDNA.
25
Table 4. List of primers used for QPCR:
Primers used Sequences
Exon 6 Forward primer 5’ GTG GTT GAA GAT GGC TAT GA 3’
Exon 6 Reverse primer 5’CTG GAA GGA ACA CAT GAG G 3’
QPP1 Endogenous Forward primer 5’ CCA GTA GAC TCC ACG ACA TAC 3’
QPP1 Reverse primer 5’ AGC CCA TCA CCA TCT TCC AG 3’
Exon 6 Forward and Reverse primers were used to detect overall PPP1CC2 mRNA levels. QPP1
Endogenous Forward and Reverse primer were used to detect endogenous mRNA levels of
PPP1CC2.
Sperm motility analysis: Sperm from caudal epididymis were extruded and collected in pre-incubated and warmed modified human tubal fluid medium, m‐HTF (Irwine Scientific) without HEPES, supplemented with BSA
o (5mg/ml). Collected sperm were then incubated for 10min at 37 C with 5% CO2 allowing them to swim out and disperse in the media. After 10min of incubation, in order to allow them to further disperse the sperm before petri dishes containing the sperm were swirled gently before measurement of motility parameters. Next, sperm were diluted to 1:20 times in m‐HTF medium and from the diluted sperm suspension 25 μl was loaded onto a chamber of 100μm Leja Chamber
2 slide. Slides were previously warmed to 37ºC using a MiniTherm stage warmer. Large bore pipet tips were used for pipetting sperm suspensions. Analysis of sperm motility on the basis of different
26
parameters was performed using CASA (Computer Assisted Sperm Analyzer) equipped with the
CEROS sperm analysis system (software version 12.3, Hamilton Thorne Biosciences, Beverly,
MA). For motility analysis, default Mouse‐2 settings from Hamilton Thorne were used with minor adjustments. The default settings were: 60frames/sec, 90 frames acquired, minimum contrast: 30, default cell size: 13 pixels, minimum cell size: 4 pixels, default cell intensity: 75, cells progressive if VAP >50 μm/sec and STR > 50%, slow cells were counted as motile, low VAP cut off: 10
μm/sec, low VSL cutoff = 0 μm/sec. For each chamber 10‐12 random non‐overlapping fields were recorded for analysis purposes. For both the control animal and STZ-line, motility was recorded independently from three animals (6weeks or older) and the values for each motility parameter were expressed as mean of the three.
Waveform Analysis All the waveform analysis were performed by Dr. Donner Babcock, University of Washington.
Stop-motion images of sperms tethered at the head to a glass chip were collected at an interval of
33ms. Next, images were analyzed using commercially available software [metamorph, Universal
Imaging, Downingtown, PA, and custom-designed software written in the igorpro (WaveMetrics,
Lake Oswego, OR) environment] (25).
27
Chapter III: RESULTS
AIM
1. Does overexpression of PP1CC1 or PP1CC2 affect spermatogenesis and sperm function?
Background and rationale: Previous studies from our lab has shown that, significant expression of PPP1CC2 protein is required to overcome infertility phenotype seen in PPP1CC null mice. Complete restoration of both fertility and spermatogenesis was noticed at PPP1CC2 protein expression levels greater than
75% of the level of PPP1CC2 expression in testis of a PPP1CC +/- control male (24). At lower levels of PPP1CC2 expression (below 75% of PPP1CC +/-), males are oligospermic, and a significant proportion of sperm formed (about 53-77%) were teratozoospermic. However, in general, the percentages of both motile sperm and progressively motile sperm increased with increasing levels of PPP1CC2 levels in testis. Thus, a threshold level of PPP1CC2 expression in testis appears to be required for normal sperm morphogenesis and spermiation (24).
In this study we wanted to determine whether overexpression of PPP1CC2, beyond that found in wild type (+/+) testis, would affect sperm function and male fertility. We thus generated mice carrying four copies of PPP1CC2 - two endogenous alleles and two transgene copies of PPP1CC2.
In addition we also produced mice expressing PP1CC1 as a transgene in a wild type background
– these mice will express both PPP1CC isoforms in testis. .
28
1.1 PPP1CC2 levels in testis of STZ (++/TgTg) and wild type (+/+) mice The objective of the aim was to check whether copies of PPP1CC2 transgene in addition to the endogenous alleles, leads to overexpression of PPP1CC2 protein in testis. Testis extracts were made in HB+ buffer and were then subjected to protein estimation. Equal protein amounts of testis extract from WT and STZ-line (++/TgTg) were subjected to western blot analysis. Visual examination and quantification of PPP1CC2 protein levels by measuring the band intensity (Fig.
12A), showed that the PPP1CC2 protein levels were the same in STZ-line (++/TgTg) and wild type (+/+) testis. Tubulin control (Fig. 12 B) was used to confirm that equal amounts of protein were loaded. Figure 12 A shows no change in PPP1CC2 protein levels in the STZ-line when compared to the Wild type control in testis.
Figure 12A : Western blot analysis of PPP1CC2 levels in testis of Wild type (+/+) and
STZ(++/TgTg). Testis of 3 month old mice were homogenised in HB+. Extracts boiled with
29
laemlli buffer were subjected to western blot analysis. 5µg of Wildtype (+/+) control,
Heterozygous for PPP1CC(+/-), Knockout for PPP1CC(-/-) and STZ-line (++/TgTg) testis extracts were loaded. Western blot analysis of testis extracts of three different animals from the
STZ-line [STZ(II)M1,M3,M4] are shown. B: Same blot was reprobed with β-tubulin antibody for loading control.
1.1.1 Densitometry analysis to determine PPP1CC2 levels in testis of Wild type (+/+) and
STZ (++/TgTg).
Animal Line Number of Animals Average Densitometric
Checked for values + SEM (Arbitary
Units)
Wild type 7 114.25 ± 3.96
STZ 11 106.75 ± 5.57
Figure 12C. Relative PPP1CC2 protein levels(in terms of percentage) in testis of Wild type
30
(+/+) and STZ (++/TgTg). The immunoreactive band intensity of PPP1CC2 in western blots were compared between wild type (+/+) and STZ (++/TgTg).Standard error of mean(SEM) was calculated and data is shown in arbitary units.
1.2 PPP1CC2 protein expression in sperm of STZ-line (++/TgTg) and Wild type (+/+) control Next we determined PPP1CC2 protein levels in mature spermatozoa from Wild type(+/+) control and STZ-line (++/TgTg) mice. Sperm was extruded from caudal epididymis in 1X PBS. After sperm count, extracts were made in 1% SDS and 6X Laemlli buffer. All sperm extracts were then adjusted to 1 x 105 sperm/μl. 10μl of the sperm count adjusted extracts, (equvalent to 1 x 106 sperms) were used in PAGE followed by western blot. Intensities of band were then compared with that of Wild type control by densitometry analysis.
PPP1CC2 protein levels, as indicated by Western Blot band intensity, in the STZ-line(++/TgTg) were equivalent to PPP1CC2 protein amounts in Wild type sperm. (Figure 13 A) Tubulin labelling
(Fig. 13 B) was used to confirm that equal amount of protein was loaded.
31
Figure 13A: Western blot analysis of PPP1CC2 levels in sperm of Wild type (+/+) control and STZ (++/TgTg). Whole sperm extracts from Wild type controls (+/+) ,Heterozygous for
PPP1CC (+/-), and STZ-line(++/TgTg) of mice aged 3 months were sperm counted and 1X106 of sperm were loaded in each lane for detection with anti-PPP1CC2 antibody. Western blot analysis of extracts of three different animals from the STZ-line [STZ (II) M1, M3, M4] have been shown.
B: The same blot was re-probed with β-tubulin for equal loading control.
1.2.1 Densitometry analysis to determine PPP1CC2 levels of sperm in Wild type (+/+) and
STZ (++/TgTg).
Mouse Lines Number of mice Average Densitometry
values + SEM (arbitary
Units)
WT 7 110 ± 4.08
STZ 11 107.75 ± 4.11
32
Figure 13C. Relative PPP1CC2 protein levels (in terms of percentage) in testis of wild type
(+/+) and STZ (++/TgTg).Standard error of mean (SEM) was calculated and data is represented in arbitrary units. The immune reactive band intensity (expression level) of PPP1CC2 were compared between Wild type (+/+) and STZ (++/TgTgPPP1CC2) .
33
1.3 PPP1CC2 mRNA levels in testis of Wild type (+/+) and STZ (++/TgTg) animals Next we wanted to determine the mRNA levels in the testis. Messenger RNA (mRNA) levels were determined in wild type and STZ mice. Primers (Table 3 and Figure 14 A) used were capable of amplifying transcripts from both the endogenous alleles and from transgenes. We found that the
PPP1CC2 transcript levels were almost double in testis of STZ mice compared to its wild type control (Figure 14C). Primers (Table 3 and Figure 14 B) were designed to quantify mRNA derived only from the endogenous alleles of Ppp1cc. Figure 14D shows that the levels of endogenous
PPP1CC2 were similar in STZ and Wild type testis (Figure 14D).
Figure 14 A: Schematic representation of design of primers used to check for total PPP1CC2 mRNA levels.
34
Figure 14 B: Schematic representation of design of primers used to check for endogenous
PPP1CC2 mRNA levels.
35
Figure 14C. Relative PPP1CC2 mRNA levels in testis (In terms of percentage) of Wild type
(+/+) and STZ (++/TgTg). Results of RT-qPCR showing overall PP1CC2 mRNA comparison between Wild type (+/+) and STZ-line (++/TgTg PPP1CC2).The mRNA expression level is shown as relative levels compared to Wild type control. The relative levels are mean of three sets of mice all of the same age. D: Relative endogenous PPP1CC2 mRNA levels in testis (in terms of percentage) of Wild type (+/+) and STZ (++/TgTg). Results of RT-qPCR showing endogenous
36
PP1CC2 mRNA comparison between Wild type (+/+) and STZ-line (++/TgTg PPP1CC2).The mRNA expression level is shown as relative levels compared to wild type control. The relative levels are mean of three sets of mice all of the same age.
1.4 Testis Weight, Sperm Number and Fertility of Wild type (+/+), STZ (++/TgTg PPP1CC2) TW (++/Tg PPP1CC1) Table 5 below shows a comparison between the testis weight and the sperm number of the different lines. Total seven animals were counted for the STZ line (++/TgTg) and the Wild type (+/+) control, while four animals were counted for TW (++/Tg PPP1CC1) line. It can be seen from the data that, the testis weight for all the three lines remain comparable (Wild type: 88.5 ± 2.59 mg,
STZ: 86.5 ± 4.4 mg, TW: 89.5 ± 5.1 mg). But, when the sperm number were compared TW-line having one copy of PPP1CC1 transgene on a wild type background, shows a reduction in their numbers (3.07± 1.066 X 107) when compared to the Wild type and STZ line, while the Wild type
(5.3 ± .173 X 107) and STZ (5.9 ± .144 X 107) sperm number were comparable(Table 5).
37
Table 5: Comparison of Testis Weight and Sperm Number between Wild type (+/+), STZ
(++/TgTg) and TW (++/Tg). The mean values obtained for each phenotype of a sample size (n)
± standard error of mean (SEM) are shown.
Mouse Lines Total Number of Testis Weight ± Sperm Count ± animals counted for SEM SEM
7 Wild type 7 88.5 ± 2.5 mg 5.3 ± .17 X 10
7 PPP1CC2 STZ 7 89.5 ± 4.4 mg 5.9 ± .14 X 10 (++/TgTg)
7 PPP1CC1 TW 4 89.5 ± 5.1 mg 3.07 ± 1.06 X 10 (++/Tg)
The fertility testing data showed that the fertility rate of STZ (++/TgTg) and Wild type (+/+) mice are comparable (Table 6). A total of 7 fertility testing was set up for the STZ-line and all 7 tests were found to be positive, with an average litter size of 11. For the TW line (one copy of PPP1CC1 on a wild type background), fertility testing data has revealed them to be sub-fertile. This may be due to the lack of regulation of PP1, and due to overexpression of PPP1CC1 over PPP1CC2 background (22).
38
Table 6: Fertility data of Wild type (+/+) and STZ (++/TgTg).
Mouse Lines Total fertility Fertile Infertile Average Fertility tested Litter size status range Wild type 11 11 0 10-14 Fertile
PPP1CC2 7 7 0 9-13 Fertile (++/TgTg)
1.5 Analysis of sperm motility Wild type (+/+) and STZ-line (++/TgTg)
Figure 15A. Total Percentage motility and Progressive motility of sperms in Wild type (+/+) and STZ (++/TgTg). Computer assisted sperm analysis of freshly prepared mature caudal epididymal spermatozoa was performed on sperm from adult (8‐12 weeks old) mice from wild
39
type control and STZ-line. Both total percent motile and progressive motility showed no significant difference. Sperm with a velocity greater than 50µm/sec were considered to be progressively motile
Total motility of wild type and the STZ-line, as well as progressive motility were found to be similar. While the total percentage of motile sperm for the wild type was at 81 ± 4.67 %, the total percentage of motile sperm for Wild type was at 82.67 ±7.31 %. The total percentage of progressively motile sperm was at 58 ± 4.87 % for the wild type, the percentage was similar and comparable to the STZ-line at 61 ± 6.21 %. While the total percentage motility and progressive motility was seen to be reduced in the TW-line (work done by my colleague Tejasvi Dudiki) having one copy of PPP1CC1 transgene on a wild type background (22).
Figure 15B. Flagellar beat waveform analysis of Heterozygous (+/-), STZ (++/TgTg
PPP1CC2) and TW (++/Tg PPP1CC1). Sperm motility was recorded on DVC high speed camera
40
with 2 msec exposure to obtain recording speeds of 100 frames/sec. Sperm flagellar beat of a single sperm from these recordings were traced and shown above. The Heterozygous (PPP1cc +/-
) sperm show flagellar beat symbolic of progressive motility with an amplitude of 20.9µm (22) and was similar and comparable to the STZ-line (++/TgTgPP1CC2) sperm motility 28µm.
However the TW-line (++/TgPPP1CC1) sperm show significantly decreased amplitude of 12.6µm
(22). The tracings were provided by Dr. Donner Babcock.
It has already been mentioned above, that the TW-line has been found to be sub fertile. So, the motility of the STZ-line was compared with TW-line and a Hetero (+/-) control (22). It was seen that the beat amplitude measured at 30µm along the beat axis, to be reduced drastically from 28
µm in the STZ-line to 12.6 µm for the TW-line (22) (Figure 15B). In contrast, the resting flagellar beat frequency was drastically reduced in TW-line (22) and showed a lack of progressive motility when compared to that of the STZ-line (Figure 15B). This drastic reduction of beat amplitude and motility might be the major cause of sub-fertility of the TW-line (22). Thus, re-emphasizing on the requirement of optimal amounts of PPP1CC2 protein, for maintaining viable fertility rates.
41
1.6 TW-line (++/Tg PPP1CC1) showed a reduction in PPP1CC2 protein levels in sperm in comparison to STZ-line (++/TgTg PPP1CC2), and Wild type control
Whole sperm extracts from TW (++/Tg PPP1CC1), wild type control (+/+) and STZ-line
(++/TgTg) were used for western blot analysis, to check for PPP1CC2 expression. After extruding sperm from caudal epididymis, normalizing the sperm count to 1X105/μl and making the extract in 1% SDS and 6X sample buffer, 1 X 106 sperm were loaded for each sample.
After visual estimation and quantification of PPP1CC2 protein levels by measuring the band intensity (Fig. 16A and 16D), it was found that the levels of PPP1CC2 were reduced in TW (++/Tg
PPP1CC1) when compared to Wild type (+/-) and STZ (++/TgTg PPP1CC2) sperm. Thus, suggesting that, the subfertility of the TW-line (22) may be due to the reduced level of PPP1CC2.
42
Figure 16 A. Western blot analysis of PPP1CC2 protein expression in sperm of Wild type
(+/+), TW (++/PPP1CC1Tg) and STZ (++/PPP1CC2TgTg). Whole sperm extracts from Wild type controls (+/+), STZ-line (++/TgTg PPP1CC2), T-line (--/Tg PPP1CC1, Rescue for
PPP1CC1)(22)and TW-line (++/Tg PPP1CC1) of mice aged 3 months were sperm counted and
1X106 of sperm were loaded in each lane for detection with anti-PPP1CC2 antibody. T-line sperm extract was used as a negative control to show the specificity of anti-PPP1CC2 antibody. B: The same blot was re-probed with β-tubulin for equal loading control. C: Showing presence of
PPP1CC1 protein in sperm in TW-line (++/Tg PPP1CC1) and T-line (--/Tg PPP1CC1), and lack of it in Wild type (+/+) and STZ-line (++/TgTg PPP1CC2) sperm, as expected, when probed with anti-PPP1CC1 antibody.
43
1.6.1 Densitometry analysis to determine the PPP1CC2 levels in sperm of Wild type (+/+),
STZ (++/TgTg PPP1CC2) and TW (++/Tg PPP1CC1).
Animal Lines Number of animals counted Densitometry values + SEM
(Arbitrary Units)
Wild type 7 110 ± 4.08
STZ 11 107.75 ± 4.11
TW 4 36.25 ± 12.48
Figure 16D. Relative PPP1CC2 protein levels (in terms of percentage) in sperm of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg PPP1CC1). Standard error of mean (SEM) was calculated and data is represented in arbitrary units. The immune reactive band intensity
(expression level) of PPP1CC2 were compared between wild type (+/+) and STZ
(++/TgTgPPP1CC2) .
44
1.7 Protein phosphatase 1 (PP1) activity of Wild type (+/+), STZ (++/TgTg PPP1CC2), and TW (++/Tg PPP1CC1) The aim of the experiment was to compare the levels of protein phosphatase 1 (PP1) activity between the above mentioned lines. It was seen that there was minimal difference when total PP1 activity was compared between the Wild type (+/+) control and STZ (++/TgTg PPP1CC2), both in sperm supernatant and pellet. While, the Wild type supernatant showed a total activity of
1869.32 ± 392.53 Nano mole/minute/105 sperms, the STZ line activity was at 2070.9 ± 294.72
Nano mole/minute/105 sperms, similarly, in the pellet as well, the values were comparable between the two lines. The Wild type total activity was at 3089.39 ± 349.92Nano mole/minute/105 sperms, the STZ line had an activity of 2881.4 ± 111.3192Nano mole/minute/105 sperms. The total phosphatase activity also remained comparable between the Wild type control and STZ. But, for the TW-line (++/Tg PPP1CC1), it was seen that the total PP1 activity, especially in the sperm supernatant, was found to be much higher both in comparison to the wild type control and STZ
(Figure 17C and 17D). This high level of PP1 activity might be one of the chief reasons for the
TW-line being sub-fertile (22). So, it can be concluded from the findings of this experiment that, it is the optimum level of PPP1CC2, which leads to high regulation of PP1 activity, thus, maintaining normal fertility ratio.
45
1.7.1 Total phosphatase activity of Wild type (+/+), STZ (++/TgTg PPP1CC2) and TW
(++/Tg PPP1CC1) in supernatant of sperm.
Animal Lines Number of animals checked Average Total Phosphatase
for activity(Supernatant) in Nano
mole/minute/105 sperms +
SEM
Wild type 3 2808.41 ± 340.73
STZ 3 3073.46 ± 355.73
TW 3 5019.6 ± 368. 72
Figure 17A: Total protein phosphatase activity in the soluble cytosolic sperm fraction
(Supernatant) Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms. Standard error of
46
mean (SEM) was calculated and data is represented in Nano Moles/minute/105 sperms. Total phosphatase activity represented as the combined phosphatase activity of PPP1CC and PP2A.
1.7.2 Total phosphatase activity of Wild type (+/+), STZ (++/TgTg PPP1CC2) and TW
(++/Tg PPP1CC1) in pellet of sperm.
Animal Lines Number of animals checked Average total phosphatase
for activity(Pellet) in Nano
moles/minute/105 sperms ±
SEM
WT 3 4332.56 ± 333.36
STZ 3 4386.11 ± 111.31
TW 3 5048.45 ± 473.45
Figure 17B: Total protein phosphatase activity in the insoluble cytosolic sperm fraction
(Pellet) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms. Standard error of
47
mean (SEM) was calculated and data is represented in Nano Moles/minute/105 sperms. Total phosphatase activity represented as the combined phosphatase activity of PP1CC and PP2A.
1.7.3. Protein Phosphatase 1 (PP1) activity of Wild type (+/+), STZ (++/TgTg PPP1CC2) and TW (++/Tg PPP1CC1) in supernatant of sperm.
Animal Lines Number of animals checked Average PP1
for activity(Supernatant) in
Nano moles/minute/105
sperm + SEM
Wild type 3 1869.32 ± 392.53
STZ 3 2070.9 ± 294.72
TW 3 3978.66 ± 158.86
Figure 17C: Total protein phosphatase 1 activity in the soluble cytosolic sperm fraction
(Supernatant) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms. Standard error
48
of mean (SEM) was calculated and data is represented in Nano Moles/minute/105 sperms. Total protein phosphatase activity measured by inhibiting PP2A activity with 2nM okadaic acid.
1.7.4. Protein Phosphatase 1(PP1) activity of Wild type (+/+), STZ (++/TgTg PPP1CC2) and TW (++/Tg PPP1CC1) in whole pellet of sperm.
Animal Lines Number of animals counted Average PP1 activity(Pellet)
for in Nano moles/minute/105
sperms + SEM
Wild type 3 3089.39 ± 349.92
STZ 3 2881.4 ± 111.31
TW 3 3381.68 ± 473.45
49
Figure 17D: Total protein phosphatase 1 activity in the insoluble cytosolic sperm fraction
(Pellet) of Wild type (+/+), STZ (++/TgTg) and TW (++/Tg) in sperms. Standard error of mean
(SEM) was calculated and data is represented in Nano Moles/minute/105 sperms. Total protein phosphatase activity measured by inhibiting PP2A activity with 2nM okadaic acid.
1.8 Protein Kinase A (PKA) mediated serine-threonine phosphorylation levels of substrates in sperm of Wild type (+/+), Heterozygous (+/-), STZ (++/TgTg PPP1CC2), and TW (++/Tg PPP1CC1).
It has been found previously that the activity of PKA is inversely proportional to that of PP1CC2 activity (2). We wanted to determine the PKA activity as measured by PKA phospho substrate levels. 1.5 X 106 extracts from sperm for each animal, made in 1% SDS and 6X sample buffer, was subjected to gel electrophoresis and western blotting and was probed with anti Phospho-
Protein Kinase A substrate antibody(1:1000 dilution, Rabbit Monoclonal)(32). It can be seen in figure 18 A, while PKA-mediated serine-threonine phosphorylated substrate profile in STZ is similar and comparable to the Wild type control. The TW line shows reduced serine-threonine phosphorylation mediated by PKA on its substrates, perhaps due to high PP1 activity (Figure 17
C and D).
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Figure 18A: Western blot analysis of profile of substrates phosphorylated by Protein
Kinase A (PKA) on their serine-threonine residues of Heterozygous (+/-), Wild Type (+/+),
TW (++/TgPPP1CC1) and STZ (++/TgTg PPP1CC2). Anti-phospho Protein Kinase A (PKA) substrate antibody was used to detect the phosphorylated substrates on whole sperm extracts
(1.5X105 sperm/lane) B: Same blot was re-probed with loading control Tubulin to show equal amount of sperm were loaded in each lane.
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1.9.1 Spz-1 protein expression in testis of STZ-line (++/TgTg) and Wild type (+/+) control
The main objective of these experiments were to determine whether the levels of the protein Spz1, reported to be a isoform specific binding partner of PPP1CC2 in testis, were altered in the STZ- line. Testis extracts were made in HB+ buffer and were then subjected to protein estimation by
Bradford assay. After estimation, protein adjusted amounts of testis extract from Wild type (+/+) control and test animals of the STZ-line (++/TgTg) were subjected to western blot analysis. 40μg of protein extracts were used for each animal and was run on the gel and immune detected using anti-Spz-1 antibody. Intensities of band were then measured for Wild type control and STZ sperm by visual inspection and by densitometry.
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Figure 19A: Western blot analysis of Spz-1 levels in testis of Wild type (+/+) and STZ
(++/TgTg).Testis of 3 month old mice were homogenized in HB+ and the extracts were protein estimated by Bradford assay. Then extracts boiled with laemlli buffer and were subjected to western blot analysis. 40µg of Wild type (+/+) and STZ-line (++/TgTg) extracts were loaded onto the gel and was probed with anti-Spz-1 antibody. B: Same blot was re probed with anti-
PPP1CC2 antibody to check levels of PPP1CC2 expression, which were found to be equal. C:
Same blot was re probed with β-tubulin antibody for loading control.
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1.9.2 Densitometry analysis to determine Spz-1 levels in testis of Wild type and STZ
Animal Lines Number of animals counted Average Densitometry value
for + SEM (Arbitary units)
WT 7 114.25 ± 3.96
STZ 3 161.67 ± 3.82
Figure 19D: Relative Spz-1 protein levels(in terms of percentage) in testis of Wild type
(+/+) and STZ (++/TgTg). Standard error of mean (SEM) was calculated and data is represented in arbitrary units. The immunoreactive band intensity (expression level) of Spz-1 were compared between Wild type ( +/+) and STZ (++/TgTgPPP1CC2).
It can be seen that the levels of spermatogenic zip protein -1 (Spz1) are higher (161.67 ± 3.82) in
STZ compared to wild type (161.67 ± 3.82) (Figure 19A and 19B). The same blot was re-probed
(Figure 18D) to check the levels of PPP1CC2 protein, and the levels were found to be equal.
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1.9.3 PPP1R11 (I3) protein expression in testis of STZ-line (++/TgTg) and Wild type (+/+) control It has been reported previously that PPP1R11 (Protein phosphatase regulatory subunit 11) is a potent inhibitor of PP1 (27). So, the thinking behind the experiment was to find out that, since,
PPP1CC2 protein levels are maintained at an optimal amount in the STZ-line, it might be possible that the increased expression of PPP1R11 might in turn inhibit PPP1CC2.
Figure 20A: Western blot analysis of PPP1R11 levels in testis of Wild type (+/+) and STZ
(++/TgTg). Testes of 3 month old mice were homogenized in HB+ and the extracts were protein estimated by using Bradford assay. Then extracts boiled with lamelli buffer were subjected to western blot analysis. 5µg of Wild type (+/+), Heterozygous for PPP1CC (+/-), Knockout for
PPP1CC (-/-) and STZ-line (++/TgTg) extracts were loaded in each lane and was probed with anti-PPP1R11 antibody. Western blot analysis of testis extracts of three different animals from the STZ-line [STZ (II) M1, M3, M4] have been shown.
It was found by using densitometry that the levels of PPP1R11 in the STZ-line to be considerably higher (140.67 ± 2.02) when compared to the wild type (114.25 ± 3.96) (Figure
20A and 20B).
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1.9.4. Densitometry analysis to determine PPP1R11 levels in testis of Wild type (+/+) and
STZ (++/TgTg).
Animal Lines Number of animals counted Average Densitometry value
for + SEM (Arbitrary Units)
Wild type 7 114.25 ± 3.96
STZ 3 140.67 ± 2.02
Figure 20B: Relative PPP1R11 protein levels (in terms of percentage) in testis of Wild type
(+/+) and STZ (++/TgTg). Standard error of mean (SEM) was calculated and data is represented in arbitrary units. The immune reactive band intensity (expression level) of PPP1R11 were compared between wild type (PPP1CC +/+) and STZ (++/TgTgPPP1CC2).
56
SUMMARY: The main objective was to check whether overexpression of PPP1CC2 affected spermatogenesis and sperm function. It was found that, PPP1CC2 levels were the same in testis and sperm from the
STZ-line (++/TgTg PPP1CC2) and compared to wild type (+/+) mice. But, in the TW-line (++/Tg
PPP1CC1) there was a significant reduction in the levels of PPP1CC2 in sperm when compared to
Wild type control and STZ. The total PPP1CC2 mRNA levels for the STZ-line showed a doubling in its levels, when compared to the Wild type control. Total protein phosphatase 1 (PP1) activity was also unaltered in sperm from STZ mice compared to wild type mice. In contrast, in sperm containing both PPP1CC1 and PPP1CC2, Protein Phosphatase 1 catalytic activity was higher when compared to wild type sperm. Protein Kinase-A mediated serine threonine phosphorylation also seems was reduced in sperm from TW-line, where PKA phosphorylation was unaltered in sperm from wild type and STZ-lines. From previous studies, it is known, that high level of Spz-1 inhibits
PPP1CC2 activity. Our data shows an increase in Spz-1 protein levels. Further studies are need to determine how this increase in levels of Spz-1 along with the increase PPP1R11 could be involved in the regulation of PPP1CC levels in testis.
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Chapter IV
DISCUSSION:
A. Optimal levels and maintenance of PPP1CC2 protein is important for maintaining normal spermatogenesis, sperm function and fertility rates.
These data suggest that PPP1CC2 protein amount are regulated in testis and sperm (Figure 12A and 13A). From visual estimation and densitometry, it was seen, when two copies of PPP1CC2 transgene is incorporated on a wild type background, PPP1CC2 showed almost no increase in its protein levels, PPP1CC2 protein levels were similar and comparable to the wild type control.
It was also found that, there seems to be an increase in overall PP1 message levels, in the STZ-line
(++/TgTg PPP1CC2), having two copies of PPP1CC2 transgene on wild type background, when compared to a Wild type (+/+) (Figure 14A). When the endogenous message levels of PPP1CC2 were compared, the STZ-line message levels were similar and comparable to that of the Wild type control (Figure 14B).
Moreover, it has also been found that levels of PPP1CC2 protein in sperm is lesser in the TW-line
(++/Tg PPP1CC1) when compared to Wild type control and STZ (Figure 16A).This data suggest that the maintenance of PPP1CC2 at optimum levels is of utmost importance for maintaining spermatogenesis, sperm function and fertility. This regulatory mechanism was inefficient/ circumvented if PP1CC1 is expressed along with PP1CC2 in testis that resulted in compromised male fertility (22). Sperm of these mice incorporate both PP1CC1 and PP1CC2 but PP1γ2 levels
58
are down regulated, as found in the TW-line, overexpressing one copy of ubiquitous PP1 isoform
PPP1CC1 on a wild type background, lends it sub-fertile (22). Lack of regulation of PPP1CC2 might be the major reason for its sub-fertility. The findings clearly reveals, that optimal levels of
PPP1CC2 proteins maintained due to the high levels of regulation of PPP1CC2 is an important determining factor, for normal fertility rates, along with proper sperm function. Our study indicates that there is not only need for maintenance of strict lower threshold requirement of PPP1CC2 protein levels but also a need for maintenance of upper limit.
B. High levels of regulation of PP1 activity is of utmost importance for maintaining normal sperm function and fertility rates
Along with optimum levels of PPP1CC2, PP1 activity also gets highly regulated. It has been seen, that, the levels of PP1 activity is highly comparable and similar between the STZ-line, having two copies of PPP1CC2 transgene on a wild type background and the wild type. Both, sperm supernatant and pellet PP1 activity were measured, and the levels were found to be similar between the STZ-line and Wild type control, indicating a high level of PP1 activity regulation. But, in the
TW-line, overexpressing one copy of ubiquitous PP1 isoform PPP1CC1 on a wild type background, the PP1 activity was found to be significantly higher, when compared both to the
Wild type control as well as the STZ-line. The PP1 activity was found to be significantly higher especially, in the sperm supernatant compared to Wild type and STZ-line (Figure 16A and B).
Thus, it can be predicted, that the high levels overall PP1 activity, which is seen in the TW-line, t, lends it sub-fertile (22).
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C. Lack of Protein Kinase A (PKA) mediated serine-threonine phosphorylation might be the major cause for the TW (++/Tg PPP1CC1) line being sub-fertile.
It has been found previously seen, that the activity of PKA is inversely proportional to that of
PP1CC activity (2). So, it was hypothesized that, PKA mediated serine- threonine phosphorylation profile for the STZ-line (++/TgTg PPP1CC2) will also be similar comparable to a Wild type (+/+).
It was found, that PKA-mediated serine-threonine phosphorylation of its substrates was similar for
STZ and Wild type control. The TW line shows a lack of serine-threonine mediated phosphorylation by PKA, attributed to the high PP1 activity, due to, the overexpression of ubiquitous isoform of PPP1CC1 on a wild type background (Figure 17A). This lack of serine- threonine phosphorylation by PKA might be one of the reasons for the TW line to sub-fertile and lack of sperm motility, and making PPP1CC2 unique and indispensable as seen in STZ, in maintaining normal spermatogenesis and sperm function. This lack of serine-threonine phosphorylation by PKA might be one of the reasons for the TW line to be sub-fertile (22), and the reason maintenance of PPP1CC2 at a particular level, as seen in STZ, essential for normal spermatogenesis and sperm function.
D. Increased expression of Spermatogenic Zip protein -1 (Spz-1) might lead to regulation of PPP1CC2
The mechanism by which PPP1CC2 expression is regulated is unknown, but we speculate that
Spz1 might be a crucial component. It has been shown that Spz1 binds to PPP1CC2 in an isoform specific manner by its unique carboxy-terminal tail. We speculate that PPP1CC2 auto‐regulates its
60
own activity by binding to Spz1 (26). It has been previously reported that increasing concentrations of Spz-1 has led to inhibition of PPP1CC2 activity. Going by the hypothesis, Spz-1 levels were compared between Wild type control and STZ-line.
From estimation and densitometry analysis, it was found that the levels of Spz-1 were considerably higher in the STZ-line compared to its Wild type control. Thus suggesting that higher level of Spz-
1 might be leading to regulation of PPP1CC2.
E. Protein phosphatase regulatory subunit 11 (PPP1R11) might also regulate PPP1CC2 A large number of PP1 interacting proteins have been identified in a wide range of somatic tissues and organisms, of which PPP1r7 (Sds22), PPP1R11 (I‐3) and PPP1R2 (I‐2) are the only PP1 regulators identified in spermatozoa. It is intriguing that the regulators of the mammal and sperm‐ specific isoform PPP1CC2 are ubiquitously expressed and are among the evolutionarily ancient
PP1 regulators. It has been reported previously that PPP1R11 (Protein phosphatase regulatory subunit 11) is a potent inhibitor of PP1. PPP1R11 protein levels are also reciprocally related to the testis specific isoform of PP1 i.e. PPP1CC2 (27). So, when PPP1R11 levels were compare between Wild type control and STZ-line, it was seen that the levels of the inhibitor was higher in the STZ-line in comparison to the Wild type. This data partially reveals the fact that higher levels
PPP1R11 protein, already a known potent inhibitor PPP1CC2, might result, in regulating the expression of PPP1CC2. The mechanism by which PPP1R11 regulates PPP1CC2 is still unknown.
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Bibliography 1. Clermont, Yves. "Quantitative Analysis of Spermatogenesis of the Rat: A Revised Model
for the Renewal of Spermatogonia." American Journal of Anatomy 111.2 (1962):111-29.
2. Knobil E & Neill JD (1994) The Physiology of reproduction (Raven Press, New York)
2nd Ed.
3. Johnson, Martin H.; Everitt, Barry J.; Johnson (2000) Essential Reproduction 5th(Blackwell
Science, Malden, MA, 2000)
4. Darszon, A., T. Nishigaki, C. Beltran, and C. L. Trevino. "Calcium Channels in the
Development, Maturation, and Function of Spermatozoa." Physiological Reviews 91.4
(2011): 1305-355.
5. Clermont, Yves. "The Cycle of the Seminiferous Epithelium in Man." American Journal
of Anatomy 112.1 (1963): 35-51.
6. Russell, Lonnie D. "Sertoli-germ Cell Interrelations: A Review." Gamete Research 3.2
(1980): 179-202.
7. Russell, Lonnie Dee, and Michael D. Griswold, eds. The Sertoli Cell. Clearwater, FL:
Cache River, 1993.
8. Eddy EM (2002) Male germ cell gene expression. Recent Prog Horm Res 57:103‐128.
62
9. Kistler MK, Sassone‐Corsi P, & Kistler WS (1994) Identification of a functional cyclic
adenosine 3',5'‐monophosphate response element in the 5'‐flanking region of the gene for
transition protein 1 (TP1), a basic chromosomal protein of mammalian spermatids. Biol
Reprod 51(6):1322‐1329.
10. Zhou Y, Overbeek PA, & Bernstein KE (1996) Tissue specific expression of testis
angiotensin converting enzyme is not determined by the ‐32 nonconsensus TATA motif.
Biochem Biophys Res Commun 223(1):48‐53.
11. Macho B, et al. (2002) CREM‐dependent transcription in male germ cells controlledby a
kinesin. Science 298(5602):2388‐2390.155
12. Monaco L, et al. (2004) Specialized rules of gene transcription in male germ cells: the
CREM paradigm. Int J Androl 27(6):322‐327.
13. Hrabchak C & Varmuza S (2004) Identification of the spermatogenic zip protein Spz1 as
a putative protein phosphatase‐1 (PP1) regulatory protein that specifically binds the
PP1cgamma2 splice variant in mouse testis. J Biol Chem 279(35):37079‐37086.
14. Toscani A, et al. (1997) Arrest of spermatogenesis and defective breast development in
mice lacking A‐myb. Nature 386(6626):713‐717.
15. Wang, Eric T., Rickard Sandberg, Shujun Luo, Irina Khrebtukova, Lu Zhang, Christine
Mayr, Stephen F. Kingsmore, Gary P. Schroth, and Christopher B. Burge. "Alternative
Isoform Regulation in Human Tissue Transcriptomes." Nature 456.7221 (2008): 470-76.
63
16. Kleene KC, Distel RJ, & Hecht NB (1984) Translational regulation and deadenylation of
a protamine mRNA during spermiogenesis in the mouse. Dev Biol 105(1):71‐79.
17. Tulsani, DPR, HT Zeng, and A. Abou-Haila. "Biology of Sperm Capacitation: Evidence
for Multiple Signalling Pathways." Society of Reproduction and Fertility 63 (2007): 257-
72.
18. Vijayaraghavan, S., R. Chakrabarti, and K. Myers. "Regulation of Sperm Function by
Protein Phosphatase PP1γ2." Society of Reproduction and Fertility 63 (2007): 111-22.
19. Fardilha, M., SL Esteves, L. Korrodi-Gregório, S. Pelech, OA Da Cruz E Silva, and E.
Da Cruz E Silva. "Protein Phosphatase 1 Complexes Modulate Sperm Motility and
Present Novel Targets for Male Infertility." Molecular Human Reproduction 17
(2011):466-77.
20. Zhang P, Strud J, Carlton E, Walter C, McCarrey J. “Multiple Elements Transcriptional
Regulation from the Human Specific PGK2 promoter in transgenic mice.” Biology of
Reproduction 60(1999): 1329-1337.
21. Soler D, Kadunganattil S, Ramdas S, Myers K, Roca J, Slaughter T, Pilder S,
Vijayaraghavan S. “Expression of Transgenic PPP1CC2 in the Testis of Ppp1cc-Null
Mice Rescues Spermatid Viability and Spermiation but Does Not Restore Normal Sperm
Tail Ultrastructure, Sperm Motility, or Fertility.” Biology of Reproduction 81 (2009):
343-352.
22. Dudiki T. “Serine/Threonine phosphatses: Role in spermatogenesis and sperm
64
function.” December 2014
23. Smith G, Don P, Trautman K, Da Cruz E Silva E, Greengard P, Vijayaraghavan S.
“Primate Sperm Contain Protein Phosphatase 1, a Biochemical Mediator of Motility.”
Biology of Reproduction 54(1996): 719-727.
24. Sinha, N, S Vijayaraghavan, and S Pilder. "Significant Expression Levels of Transgenic
PPP1CC2 in Testis and Sperm Are Required to Overcome the Male Infertility Phenotype
of Ppp1cc Null Mice." PloSOne 7.10 (2012). Web. 3 June 2013.
25. Nolan MA1, Babcock DF, Wennemuth G, Brown W, Burton KA, McKnight GS,
“Sperm-specific protein kinase A catalytic subunit Calpha2 orchestrates cAMP signaling
for male fertility”.Proc Natl Acad Sci U S A. 2004 Sep 14.
26. Hrabchak C, Varmuza S.” Identification of the spermatogenic zip protein Spz1 as a
putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the
PP1cgamma2 splice variant in mouse testis.” J Biol Chem. 2004.
27. Cheng L, Pilder S, Nairn AC, Ramdas S, Vijayaraghavan S.” PP1gamma2 and PPP1R11
are parts of a multimeric complex in developing testicular germ cells in which their
steady state levels are reciprocally related”. PLoS One. 2009.
28. Chakrabarti R “Role for PP1g2 in spermatogenesis and sperm morphogenesis”. March
2007.
65
29. Ramdas S “ROLE OF PP1γ2 BINDING PARTNERS IN SPERMATOGENESIS AND
SPERM FUNCTION.” December 2012.
30. Hall, John E. The Textbook of Medical Physiology. 987-0-8089-2400-5th ed.
Philadelphia: Saunders Elsevier, 2012.
31. Lu J, et al. (2008) 99th American Association for Cancer Research Annual Meeting LB-1
an of serine-threonine protein phosphatase PP2A, suppresses the growth of glioblastoma
cells in vitro and in vivo, p 5693
32. A, Nebreda AR. Inhibition of Xenopus oocyte meiotic maturation by catalytically
Inactive Protein kinase A. Proc Natl Acad Sci U S A. 2002 Apr 2; 99(7):4361-6.
66