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research-article |
Department of Biological Sciences,3 Kent State University, Kent, Ohio 44242
Department of Anatomy and Cell Biology,4 Temple University School of Medicine, Philadelphia, Pennsylvania 19140
ABSTRACT
Serine/threonine protein phosphatase 1 (PP1) consists of four ubiquitously expressed major isoforms, two of which, PP1gamma1 and PP1gamma2, are derived by alternative splicing of a single gene, Ppp1cc. PP1gamma2 is the most abundant isoform in the testis, and is a key regulator of sperm motility. Targeted disruption of the Ppp1cc gene causes male infertility in mice due to impaired spermiogenesis. This study was undertaken to determine the expression patterns of specific PP1 isoforms in testes of wild-type mice and to establish how the defects produced in Ppp1cc-null developing sperm are related to the loss of PP1gamma isoform expression. We observed that PP1gamma2 was prominently expressed in the cytoplasm of secondary spermatocytes and round spermatids as well as in elongating spermatids and testicular and epididymal spermatozoa, whereas its expression was weak or absent in spermatogonia, pachytene spermatocytes, and interstitial cells. In contrast, a high level of PP1gamma1 expression was observed in interstitial cells, whereas much weaker expression was observed in all stages of spermatogenesis. Another PP1 isoform, PP1alpha, was predominant in spermatogonia, pachytene spermatocytes, and interstitial cells. Examining the temporal expression of PP1 enzymes in testes revealed a striking postnatal increase in PP1gamma2 levels compared with other isoforms. Testicular sperm tails from Ppp1cc-null mice showed malformed mitochondrial sheaths and extra outer dense fibers in both the middle and principal pieces. These data suggest that in addition to its previously documented role in motility, PP1gamma2 is involved in sperm tail morphogenesis.
phosphatases,, sperm, spermatid, spermatogenesis, testis
Sperm formation in mammals is characterized by a well-defined sequence of mitotic and meiotic divisions, which are followed by a long period of complex morphogenetic differentiation, leading to the production of mature spermatozoa [1]. Thus, mammalian sperm development, taking place in the seminiferous tubules of the testis, can be divided conveniently into three distinct stages: proliferative, meiotic (spermatogenesis), and spermiogenic (postmeiotic differentiation and morphogenesis). In newborn mice, male germ cell precursors undergo self-renewal in the testis at Days 1–7 postpartum. The early proliferative cell divisions lead to the development of types A and B spermatogonia, the latter of which undergo premeiotic replication and enter meiosis as primary spermatocytes. Secondary spermatocytes proceed through a brief second meiotic division in which haploid spermatids are generated. During spermiogenesis these haploid round spermatids differentiate to form elongated spermatids and, finally, mature spermatozoa over a period of approximately 2 wk. The first wave of spermatogenesis is followed by additional waves, enabling continuous sperm production throughout the life of the animal [1, 2]. Immotile testicular spermatozoa acquire the capacity for motility during transit through the epididymis [3]. Thus, spermatozoa isolated from the caput region of the epididymis are morphologically mature but immotile, whereas sperm from the caudal region of the epididymis display vigorous motility and forward progression.
Protein phosphorylation and dephosphorylation is a universal mechanism regulating metabolic functions in tissues [4, 5]. The serine/threonine phosphatase, protein phosphatase 1 (PP1), is represented by a highly conserved family of proteins in all eukaryotes. It controls a variety of processes, such as cell division, transcription, translation, muscle contraction, glycogen and lipid metabolism, sperm motility (and possibly other sperm functions), and neuronal signaling [6]. PP1 is also involved in embryonic development in fish [7]. The four mammalian isoforms of PP1, which are over 98% identical excluding their unique C-termini, are PP1
, PP1ß, PP1
, and PP12. The isoforms PP11 and PP12 are alternative splice variants of a single gene, Ppp1cc [8, 9]. Targeted disruption of Ppp1cc, resulting in the loss of PP11 and PP12, causes infertility in male mice due to impaired spermiogenesis, leading to the absence of epididymal spermatozoa [10, 11]. This indicates that one or both of the isoforms are involved in sperm development and possibly spermiation (the release of mature sperm from the seminiferous epithelium into the lumen).
While previous reports have shown that PP12 is the predominant PP1 isoform expressed in the testis [12, 13], its distribution within specific cell types in the mouse testis relative to the other PP1 isoforms is unknown. In this report isoform-specific antibodies raised against PP11, PP12, and PP1 were employed to determine the distribution of these proteins in wild-type testicular cells and spermatozoa from mice. In order to further elucidate the relative involvement of these PP1 isoforms in impaired spermatogenesis of Ppp1cc-null mice, we have analyzed the morphology of developing spermatids from Ppp1cc-null mice and compared the quantity and distribution of a variety of postmeiotically expressed proteins in wild-type testes to those aspects of postmeiotic protein expression in Ppp1cc-null testes. Our results indicate that PP12, with its unique distribution in postmeiotic germ cells and its status as the only isoform of PP1 detected in sperm, is most likely playing a key role in sperm morphogenesis.
Testes were collected from 3- to 4-mo old CD1 mice (Charles River, Wilmington, MA) or Ppp1cc-null mice. Testes were obtained from CD1 animals 8, 14, 18, and 30 (adult) days old for the study of postnatal expression. Founders for the Ppp1cc-null mice were obtained from Dr. Varmuza, Department of Zoology, University of Toronto, ON, Canada [10]. All procedures involving animals used in this study were approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee and the Kent State University Animal Care and Use Committee.
The PP11 antibody (1:5000 for Western blot [WB]; 1:200 for immunohistochemistry [IHC]) was prepared using a synthetic peptide corresponding to the 13 amino acids at the carboxy terminus of PP11 as the antigen. PP12 antibody (1:5000 for WB; 1:500 for IHC) was prepared using a synthetic peptide corresponding to the 22 amino acids at the carboxy terminus of PP12 as the antigen. The ability of this antibody to recognize PP12 is well documented [12, 14, 15]. PP1 (1:5000 for WB; 1:250 for IHC) was a generous gift from Dr. Edgar de Cruz e Silva, Signal Transduction Laboratory, Center for Cell Biology, University of Aveiro, Aveiro, Portugal [13]. Antibodies against sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7 [PPP1R7]; 1:2000 for WB; 1:400 for IHC), was made against a synthetic peptide corresponding to amino acid residues 329–342. The ability of this antibody to recognize sds22 in WB and IHC has been documented [14, 16]. A polyclonal antibody to 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) was purchased (Zymed Laboratories) and was generated using a synthetic peptide corresponding to the 20 amino acids at the N-terminus of the human 14-3-3 beta/alpha protein. This sequence is identical in the delta/zeta isoforms. AKAP4 (A kinase anchoring protein 4) antibody (1:1000 for WB; 1:500 for IHC) was prepared using a synthetic peptide corresponding to amino acids 191–204 from mouse AKAP4 and was a generous gift from Dr. E.M. Eddy, National Institute of Environmental Health Sciences, Research Triangle Park, NC [17]. Outer dense fiber 2 (Odf2) antiserum (1:1000 for WB; 1:300 for IHC) was a gift from Dr. Frans A. van der Hoorn, Department of Biochemistry and Molecular Biology, University of Calgary, AB, Canada [18]. Testis-specific cytochrome c antibody (1:1000 for WB) was a gift from Dr. J. Millán, The Burnham Institute, La Jolla, CA [19]. AKAP110/AKAP3 (A kinase anchor protein 3; 1:2000 for WB), sperm surface protein SP17 (sperm autoantigenic protein 17; 1:2000 for WB), FSII (fibrousheathin II; 1:2000 for WB), and SPNR (spermatid perinuclear ribonucleic acid binding protein; 1:2000 for WB) [20] antibodies were the gift of Dr. Daniel Carr, Department of Medicine, Oregon Health and Sciences University and Veterans Affairs Medical Center, Portland, OR [21].
Preparation of Mouse Testis and Soluble Sperm Extracts
Testes from wild-type and Ppp1cc-null mice were homogenized in RIPA lysis buffer (Upstate Biotechnology), containing 10 mM benzamidine-HCl, 1 mM PMSF, 0.01 mM tosyl phenylalanyl chloromethyl ketone (TPCK), and 5 mM ß-mercaptoethanol using 1 ml buffer for 0.1 g tissue. The homogenates were centrifuged at 16 000 x g at 4°C for 10 min. The supernatants are referred to as testis extract. Testis extracts were supplemented with 10% (v/v) glycerol and stored at –20°C until further use. Caudal epididymal spermatozoa were isolated from mouse epididymides and washed twice with Whittingham media (99.3 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 0.36 mM NaH2PO4, 25 mM NaHCO3, 25 mM sodium lactate, 0.50 mM sodium pyruvate, 5.55 mM glucose, 100 U/ml penicillin-G potassium salt, and 50 mg/ml streptomycin sulfate). Spermatozoa were collected by centrifugation, and the pelleted spermatozoa were suspended in RIPA lysis buffer containing 10 mM benzamidine-HCl, 1 mM PMSF, 0.01 mM TPCK, and 5 mM ß-mercaptoethanol. The sperm suspension was sonicated with three 5-sec bursts of a Biosonic sonicator (Bronwell Scientific, Rochester, NY) at maximum setting. The sperm sonicate was centrifuged at 16 000 x g at 4°C for 10 min. The 16 000 x g supernatants were supplemented with 10% (v/v) glycerol and stored at –20°C until further use. This preparation is referred to as soluble sperm extracts in this article.
Testis and sperm extracts for WB analysis were prepared by boiling with SDS sample buffer for 5 min. Testis and sperm proteins were separated by 12% SDS-PAGE based on the protocol of Laemmli [22]. Proteins then were electrophoretically transferred to Immobilon-P PVDF membrane (Millipore Corp., Bedford, MA). Nonspecific protein binding sites on the membrane were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.4, and 150 mM NaCl). The blots then were incubated with primary antibodies at 4°C overnight, except SP17 and FSII, which were incubated for 1 h at room temperature. After the wash, the blots were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:2000) (GE Amersham) for 1 h at room temperature. Blots were washed with TBS containing 0.1% Tween 20 at 2 x 15 min each and 4 x 5 min each. The blots then were developed with enhanced chemiluminescence.
Immunohistochemistry of Mouse Testis
Testes of both wild-type and Ppp1cc-null mice were fixed in 4% paraformaldehyde in PBS at 4°C for 40 h. The testes then were transferred to 75% ethanol and dehydrated, permeabilized, and embedded in paraffin using a Shandon Tissue Processor (Thermo Electron Corp., Waltham, MA). Multiple 8-µm-thick sections of the whole testis were attached to poly-L-lysine-coated slides, deparaffinized, and rehydrated using a standard procedure. Antigen retrieval was performed using 1x Antigen Retrieval Citra Solution (BioGenex, San Ramon, CA). Sections immersed in Citra solution were microwaved for three separate 3-min periods on high setting, with a cooling period of 1 min between each heating cycle. Sections were washed briefly in distilled water and then washed three times for 10 min with PBS. Sections were incubated for 1 h at room temperature in a blocking solution containing 10% normal goat serum in PBS. The slides then were incubated with primary antibodies for 2 h at room temperature or overnight at 4°C, washed three times with PBS, and incubated with corresponding secondary antibody (1:250) conjugated to indocarbocyanine (Cy3; Jackson Laboratories, West Grove, PA) for 1 h at room temperature. The slides were washed five times with PBS, mounted with Vectashield (Vector Laboratories, Burlingame, CA) mounting media, and examined using a FluoView 500 Confocal Fluorescence Microscope (Olympus, Melville, NY). Control slides were processed in the same manner, except that the primary antibody was omitted.
Testicular Sperm Isolation and MitoTracker Green Staining
Testicular sperm were isolated using the protocol described by Kotaja et al. [23]. In brief, testes from both wild-type and null mice were decapsulated in PBS. Seminiferous tubules were untangled manually using fine forceps, and small pieces of the tubule were stretched and observed by transillumination. Dark regions containing mature sperm were then teased open, and sperm suspensions were observed using confocal microscopy. For staining with MitoTracker Green (Invitrogen), sperm suspensions were incubated with 200 µl MitoTracker Green (0.25 mM) for 10 min and then examined using a FluoView 500 Confocal Fluorescence Microscope.
Light and Transmission Electron Microscopy
Tissue preparation for light and electron microscopy involved a modification of the technique described in Orth and Christensen [24]. Briefly, 20-wk-old mice were anesthetized with Avertin (1.25% tribromethanol injected i.p. at 25–30 µl/g body weight; Sigma-Aldrich), and then perfused through the descending aorta with 2.5% glutaraldehyde in 0.1 M sodium cacodylate. Following fixation, the testes were removed, minced, and immersed in the same fixative for 15 min at 4°C. After washing in 0.1 M sodium cacodylate buffer, the tissue blocks were postfixed in 1.33% OsO4 for 90 min at 4°C, then dehydrated through a graded series of ethanols and infiltrated with and subsequently embedded in Epon/Araldite (Structure Probe, Inc./SPI Supplies, West Chester, PA).
For light microscopy of electron microscopy-fixed tissue, 1-µm-thick sections were cut and dried onto slides, followed by staining in 1% toluidine blue in 1% sodium borate. Sections were viewed and photographed with a Leitz Orthoplan 2 (Leica Microsystems Inc.).
For electron microscopy, ultrathin sections 85 nm thick were cut and placed onto grids, followed by staining for 5 min in 10% uranyl acetate in methanol and then in Reynold lead citrate for 2 min. Sections were viewed and photographed with a Philips 400 transmission electron microscope.
Testicular Sperm Immunocytochemistry
Testicular sperm recovered from both testes of wild-type and Ppp1cc-null mice were incubated in 200 µl of 100-mM sucrose. The sperm suspension then was mounted on 1.5% paraformaldehyde-dipped slides and dried in a humidified chamber. After a brief wash in PBS, sperm were treated with 0.2% Triton X-100 in PBS. The slides then were incubated for 1 h with blocking solution containing 5% goat serum in PBS, followed by incubation in primary antibody for 2 h at room temperature or overnight at 4°C. After incubation with the primary antibody, the slides were washed three times with PBS and incubated with a secondary antibody conjugated to indocarbocyanine (Cy3) for 1 h at room temperature. The slides were washed five times with PBS, mounted with Vectashield mounting media, and examined using a FluoView 500 Confocal Fluorescence Microscope.
Expression of PP12 Increases with Age in Mouse Testis
We first determined that peptide antibodies generated against PP1, PP11, and PP12 were specific to each isoform. The amino acid sequences for the three isoforms of PP1 allowed us to select specific peptides to be used to produce isoform-specific antibodies. The C-terminal regions of PP1, PP12, and PP11 were chosen, since they exhibited the greatest divergence [7, 9]. Figure 1A shows the alignment of their deduced C-termini and highlights the peptides chosen for antibody production. WB analysis using recombinant proteins showed that PP11 and PP12 antibodies reacted specifically and strongly against the isoforms to which they were raised and showed essentially no cross-reactivity to the other isoforms (Fig. 1B). Specificity of PP1 antibody is well established [13].
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First, WB analyses were performed to determine whether all three isoforms of PP1 (PP11, PP12, and PP1) are present in mouse testis and sperm. All three PP1 isoforms were observed in testis extracts (Fig. 1C); however, PP12 was the only PP1 isoform detected in sperm extracts (Fig. 1C). These observations with mouse sperm are similar to those found with bovine, human, and rhesus monkey spermatozoa [12].
Next, we determined the expression of PP1 isoforms in testes from mice of increasing postnatal ages (Fig. 2). WB analysis was performed on extracts prepared from testis from 8-day-, 14-day-, and 18-day-old and 1- to 2-mo-old mice in each experiment. These ages were selected because they mark the approximate times at which distinct germ cell populations are produced in the testis [25]. Postnatally, from Day 1 to Day 8, spermatogonia are the only germ cells in the seminiferous tubules in the testis. From Day 8 to Day 10 there is onset of meiosis, resulting in the formation of spermatocytes. Secondary spermatocytes are formed by Day 14, and spermatid formation occurs at approximately Day 18. Postnatal expression of the different PP1 isoforms in testis was strikingly age dependent. An increase in expression of immunoreactive PP12 was observed at each of the time points examined (Days 8, 14, 18, and 30). This increase in PP12 expression is correlated with the formation of spermatocytes and spermatids in the developing testes. In contrast, expression of PP11 and PP1 appeared to decrease with postnatal age.
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Cellular Localization of PP1 Isoforms in Mouse Testis
Formaldehyde-fixed testis sections were incubated with PP11, PP12, and PP1 antibodies followed by incubation with Cy3-labeled secondary antibodies. Fluorescence patterns showed a distinct expression blueprint of the different PP1 isoforms in testis sections. PP12 immunofluorescence was predominant in the cytoplasm of secondary spermatocytes and round spermatids, elongated spermatidsm and spermatozoa at all stages of adult mouse testis (Fig. 3, A–C). Fluorescence was weak in spermatogonia, pachytene spermatocytes, and peritubular cells. Interstitial cells showed no immunoreactive PP12.
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Immunofluorescence for PP11 was distinctly different from that observed for PP12. Intense fluorescence was observed in interstitial cells (Fig. 3, D–F), and weak fluorescence was observed at all stages of spermatogenesis in adult mouse testis in both the cytoplasm and nuclei of all cells. With PP1 antibody, intense fluorescence was observed in the cytoplasm of interstitial cells, peritubular cells, spermatogonia, and pachytene spermatocytes in adult mice testis (Fig. 3, G–I). No signal was detected when secondary antibody was used alone (Fig. 3J) or with preimmune sera (Fig. 3K).
Altered Expression of PP1 in Testis of Ppp1cc-null Mouse
Ppp1cc-null male mice are infertile, apparently due to impaired spermiogenesis [10]. Here we examined whether PP1 expression and localization is altered in the testis of null mice. Expression of PP1 is high in the testis of adult Ppp1cc-null mice compared with wild-type mice testis (Fig. 4A). The localization pattern for PP1 also was altered in Ppp1cc-null (Fig. 4B) compared with wild-type testis sections (Fig. 3I). Unlike in wild-type testis sections (Fig. 3I), PP1 was present near the Sertoli cell borders and in early round spermatids in the testes of Ppp1cc-null mice (Fig. 4B). Fluorescence in spermatogonia, peritubular cells, pachytene spermatocytes, and interstitial cells was similar in wild-type and Ppp1cc-null testes. No signal was observed when secondary antibody was used alone (data not shown). As expected, PP11 and PP12 were absent in the Ppp1cc-null testis sections, further confirming the specificity of the antibodies (Fig. 4, C and D).
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Reduced Number of Spermatozoa in Testis of Ppp1cc-null Mice
We next examined the morphology of testicular cells in wild-type and Ppp1cc-null mice to further determine the nature of the spermatogenic defect in Ppp1cc-null testis. We performed histologic analysis of formaldehyde-fixed, paraffin-embedded, and hematoxylin-stained testis sections. Seminiferous tubules from Ppp1cc-null testes showed multiple vacuoles, sloughing of germ cells into the lumen, and mislocated germ cells, as previously described [10]. We also found that differentiation of postmeiotic spermatids into spermatozoa is inhibited in Ppp1cc-null mice. In agreement with a previous report [10, 11], the number of elongating spermatids in each seminiferous tubule was reduced in Ppp1cc-null (Fig. 5B) compared with wild-type (Fig. 5A) testes. However, the number of round spermatids were comparable in some of the seminiferous tubules of both wild-type and null testes. Surprisingly, epididymides of Ppp1cc-null males were virtually devoid of spermatozoa, even though Ppp1cc-null testes contained spermatozoa, albeit at lower numbers than in wild types (Fig. 5C). Instead, epididymides from Ppp1cc-null males contained immature germ cells, including round spermatids and, occasionally, pachytene spermatocytes (Fig. 5D).
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Abnormal Mitochondrial Organization, Outer Dense Fiber Complex, and Fibrous Sheath Formation in Testicular Spermatozoa of Ppp1cc-null Mice
Since the epididymides of Ppp1cc-null mice were devoid of spermatozoa, testicular spermatozoa were used to examine their morphology. Although mature sperm were reduced in number compared with normal mice, we were able to visualize sufficient mutant spermatozoa from testes of Ppp1cc-null mice to make this observation. Testicular sperm were collected using methods described by Kotaja et al. [23]. Spermatozoa were examined using confocal microscopy with differential interference contrast (DIC) optics. Instead of the wild-type hook-shaped sperm head structure (Fig. 6A), mutant sperm showed a range of head shapes, from round to oblong (Fig. 6, B, C, and F). Surprisingly, we also observed disorganized mitochondria in the midpieces of mutant sperm. In wild-type sperm, the mitochondrial sheath is helical and tightly wrapped around the midpiece (Fig. 6D). In sperm from Ppp1cc-null mice, the mitochondrial sheath was much more loosely arranged (Fig. 6F), whereas in some instances it was absent (Fig. 6I). This observation was confirmed by staining Ppp1cc -null testicular sperm with MitoTracker Green (Fig. 6, G and H).
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We also used standard light and transmission electron microscopy (TEM) of fixed sections to confirm and expand upon the observations made by confocal microscopy of Ppp1cc-null testes. Light micrographs of testis sections from Ppp1cc-null mice showed severely reduced elongated spermatid numbers near the lumen. Tubule lumens in these testes also tended to be small or absent, possibly as a result of the failure of spermatid maturation and spermiation (Fig. 7, A and B). Four distinct abnormalities were observed by TEM of testicular sperm, (Fig. 8, A–E). First, some abnormal head shapes were observed, possibly due to degenerating condensing spermatids (data not shown). More prominently, mitochondrial sheaths in the midpieces of elongating spermatids and testicular spermatozoa of Ppp1cc-null mice appeared disorganized and did not form the tightly packed helical structures (as shown by arrowheads) observed in wild-type sperm. The third and most prominently observed defect was that outer dense fibers were disorganized, with a highly increased number of florets throughout the flagella of mutant sperm. Fourth, a subtle abnormality in the development of the fibrous sheath was observed, specifically in the formation of distinct, triangular-shaped longitudinal columns and inward projections that replace the outer dense fibers associated with axonemal microtubule doublets 3 and 8 in wild-type sperm.
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Postmeiotic Protein Expression in the Male Germ Cells of Ppp1cc-null Mice
The defects in the flagella of mutant sperm prompted us to examine whether expression of a number of spermatid proteins was normal in null testis. Testis extracts from adult wild-type and Ppp1cc-null mice were subjected to WB analyses with a panel of antibodies. Testis-specific cytochrome c oxidase [19], AKAP3/AKAP110 [20], and SPNR [21], which are known to be expressed in secondary spermatocytes and spermatids, showed comparable levels of protein expression in wild-type and mutant testis extracts (Fig. 9B). However, postmeiotically expressed genes, such as fibrous sheath protein AKAP4/AKAP82, odf2, regulator of PP1 sds22, SP17, and another fibrous sheath protein (FSII) were absent or markedly reduced in Ppp1cc-null compared with wild-type testis (Fig. 9A).
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Reduced levels of proteins in adult testis as shown in WBs could reflect an insufficient number of cells where these proteins are produced. We used IHC to determine whether reduced cell number might be the cause of reduced protein levels. Immunofluorescence for AKAP4, odf2, and sds22 was observed in round spermatids and elongating spermatids in adult wild-type testis (Fig. 10, A, E, and I). Immunofluorescence for FSII was observed in the elongating spermatids of the wild-type testis sections (Fig. 10M). In contrast, in adult Ppp1cc-null testis, although round and elongating spermatids were clearly present, there was a complete lack of fluorescence for these proteins (Fig. 10, C, G, K, and O).
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Expression of AKAP4, odf2, sds22, and FSII in Testicular Sperm of Ppp1cc-null Males
To further examine the absence or reduced expression of the postmeiotic proteins in testicular sperm, we isolated testicular sperm from both wild-type and Ppp1cc-null mice and examined them by immunocytochemistry. Comparable immunofluorescences for AKAP4, odf2, FSII, and sds22 were observed in the tail in adult wild-type testicular sperm and Ppp1cc-null testicular sperm (Fig. 11). These data suggest that the reduced amount of the postmeiotic proteins seen in WBs of whole-testis extracts is due to the reduced number of the cell types in the Ppp1cc-null mice testes. Alternatively, there could be increased expression of these proteins.
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In this study we have documented for the first time the expression patterns of PP12, PP11, and PP1 in the developing postnatal mouse testis. There is a postnatal, age-dependent increase in expression of PP12 that is concomitant with an age-dependent decrease in expression of PP11 and PP1. We also have established the expression profiles of these PP1 isoforms in various cell types of the adult testis of wild-type mice, showing that PP12 is the only isoform present in abundance in secondary spermatocytes, round spermatids, and elongating spermatids, whereas for the most part, PP11 and PP1 expressions are restricted to spermatogonia, pachytene spermatocytes, and interstitial cells. Thus, the distinct expression patterns of PP1, PP11, and PP12 during postnatal testis development and subsequent rounds of spermatogenesis suggest unique male germ cell developmental functions for each of these PP1 isoforms.
In contrast to Ppp1cc-null females, all of whom are fertile, Ppp1cc-null male mice are infertile due to defective spermiogenesis, leading to an apparent failure of spermiation [10]. By detailed ultrastructural analyses using light and transmission electron microscopy, we observed numerous structural defects in elongating spermatids and testicular spermatozoa of the Ppp1cc-null male mice, including some abnormal head shapes that were in agreement with a previous report [11]. Prominent defects were poorly developed or missing mitochondrial sheaths and had supernumerary, disorganized outer dense fiber florets throughout sperm tails. We also detected frequent degeneration of condensing spermatids, as indicated by fragmentation of tail structures and the presence of numerous vacuoles in the cytoplasm of elongating spermatids, partially explaining the complete absence of spermatozoa in the epididymis of these mice. A subtle abnormality in the development of the fibrous sheath was observed, specifically in the formation of distinct, triangular-shaped longitudinal columns and inward projections that replace the outer dense fibers associated with axonemal microtubule doublets 3 and 8 in wild-type sperm. These observations suggest that PP12 is required for flagellar integrity and structures.
WB analysis and IHC of Ppp1cc-null testes also showed increased expression and altered localization of PP1 to round spermatids and to Sertoli cells at the germ cell boundary. The reason for this change in PP1 expression and localization in the absence of PP1 expression is not yet known. Next we observed the absence or sharp reduction in the levels of postmeiotic proteins (AKAP4/AKAP82, odf2, sds22, and FSII) in Ppp1cc-null testes using both WB analysis and IHC. Some of these proteins appear to be associated with sperm tail development and function [12, 14, 15, 17, 18, 20]. This reduction of protein expression could be due to a lack or reduced number of cell types expressing these proteins or reduced protein expression. Although studies using selected antibodies and immumnofluorescence in testes sections suggested reduced protein expression in null testes, further examination using immunocytochemistry of testicular sperm showed that AKAP4, odf2, FSII, and sds22 are present in mutant sperm. This suggests that the apparent reduction in intracellular protein levels seen in WB analysis could be due to a reduced proportion of cells in the testis where these proteins are synthesized.
These results suggest that in addition to its role in sperm motility regulation [12, 13], PP12 appears essential for morphogenetic processes during spermiogenesis. In its absence in Ppp1cc-null testes, spermatogenesis appears to proceed normally up until the transition from round spermatids to elongating spermatids. It is intriguing that the lack of phosphatase shows these phenotypes, whereas lack of cAMP phosphorylation in male mice lacking protein kinase A (PKA) and soluble adenyl cyclase does not show these phenotypes [26, 27]. This requirement of the PP12 isoform during spermiogenesis is surprising considering that other isoforms of PP1 substitute for PP1 in all other cells and in the female; thus, an exception is developing sperm. In this regard it may be noted that analysis of genome databases and additional studies from our laboratory show that PP12 with its unique C-terminus extension is mammalian specific. It is therefore tempting to speculate that PP12 may have an isoform-specific function in the development of outer dense fibers and the fibrous sheath, structures found predominantly in mammalian sperm. If this is true, then PP12 but not PP11 should restore sperm formation and fertility in Ppp1cc-null mice. Experiments with transgenic mice with a restricted expression of either PP11 or PP12 in testis using the Pgk2 promoter are currently underway.
In summary, our studies show unique expression of PP1, PP11, and PP12 in developing testis and male gamete formation, suggesting an independent role for each isoform in spermatogenesis. Testicular sperm tails from Ppp1cc-null mice demonstrate a variety of abnormalities in both the middle and principal pieces, suggesting roles for PP12 (whose expression increases significantly during spermiogenesis in wild-type testes) in sperm differentiation and morphogenesis.
ACKNOWLEDGMENTS
We thank Dr. Susan Varmuza, Department of Zoology, University of Toronto, ON, Canada, for providing us a breeding pair to generate Ppp1cc-null mice. We thank Kimberley Myers for her critical review of the paper. We thank Dr. Dan Carr for his generous gift of antibodies; Dr. Van der Hoorn for odf2 antibody; Dr. Millan for testis-specific cytochrome c oxidase antibody; Dr. Mitch Eddy for AKAP4 antibody; Mike Model (Department of Biological Sciences, Kent State University, Kent, OH) for his assistance in confocal fluorescence microscopy; and Ben Ingersoll (Department of Biological Sciences, Kent State University) for his assistance in animal care.
FOOTNOTES
1Supported by the National Institutes of Health (HD38520). 
Correspondence: 2FAX: 330 672 3713; e-mail: svijayar{at}kent.edu.
Received: 4 November 2006.
First decision: 6 December 2006.
Accepted: 12 February 2007.
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