Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility.

OBJECTIVE: To design protein phosphatase 1 (PP1)-disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S)/ANIMAL(S): Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effect of the bioportides on the activity and interactions of PP1γ2-a PP1 isoform expressed exclusively in testicular germ cells and sperm-and on sperm vitality and motility. RESULT(S): PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome. CONCLUSION(S): These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.

The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility.

BACKGROUND: Spermatogenesis is a complex biological process highlighted by synthesis and activation of proteins that regulate meiosis and cellular differentiation occur during spermatogenesis. 14-3-3 proteins are adaptor proteins that play critical roles in kinase signaling, especially for regulation of cell cycle and apoptosis in eukaryotic cells. There are seven isoforms of the 14-3-3 family proteins encoded by seven genes (ß, ε, γ, η, θ/τ, ζ and σ). 14-3-3 isoforms have been shown to have many interacting partners in several tissues including testis. OBJECTIVE: While it is known that 14-3-3 proteins are expressed in the functions of testis and spermatozoon, the role for each of the seven isoforms is not known. In this study, we investigated the roles of 14-3-3η and 14-3-3ε isoforms in spermatogenesis. MATERIALS AND METHODS: To study the in vivo function of 14-3-3η and 14-3-3ε in spermatogenesis, we generated testis-specific and global knockout mice for each of 14-3-3η and 14-3-3ε isoforms (CKO and GKO, respectively). Computer-assisted semen analysis was used to assess sperm motility, while immunohistochemical studies were conducted to check spermatogenesis. RESULTS: Although both 14-3-3η and 14-3-3ε isoforms were present in mouse testis, only the expression of 14-3-3ε, but not 14-3-3η, was detected in spermatozoa. Mice lacking 14-3-3η were normal and fertile while 14-3-3ε CKO and GKO males showed infertility. Low sperm count with higher abnormal spermatozoa was seen in 14-3-3ε CKO mice. The motility of 14-3-3ε knockout spermatozoa was lower than that of the control. A reduction in the phosphorylation of both glycogen synthase kinase 3 and PP1γ2 was also seen in spermatozoa from 14-3-3ε CKO mice, suggesting a specific role of 14-3-3ε in spermatogenesis, sperm motility, and fertility. DISCUSSION AND CONCLUSION: This is the first demonstration that of the seven 14-3-3 isoforms, 14-3-3ε is essential for normal sperm function and male fertility.

X-Linked Huwe1 Is Essential for Oocyte Maturation and Preimplantation Embryo Development.

HUWE1 is a HECT-domain ubiquitin E3 ligase expressed in various tissues. Although HUWE1 is known to promote degradation of the tumor suppressor p53, given a growing list of its substrates, in vivo functions of HUWE1 remain elusive. Here, we investigated the role of HUWE1 in the female reproductive system. Homozygous deletion of Huwe1 in mouse oocytes of primary follicles caused oocyte death and female infertility, whereas acute depletion of HUWE1 protein by Trim-Away technology did not impact oocytes from antral follicles. Interestingly, oocytes from Huwe1 heterozygous females matured and fertilized normally, but the majority of embryos that lacked maternal Huwe1 were arrested at the morula stage after fertilization. Consequently, Huwe1 heterozygous females only produced wild-type pups. Concomitant knockout of p53 did not recover fertility of the Huwe1 knockout females. These findings make HUWE1 a unique and critical maternal factor indispensable for maintaining the quality of oocytes and embryos.

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs.

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3ß, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.

YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation.

BACKGROUND: Immature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies. RESULTS: Messenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest. CONCLUSIONS: Multiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development.

Monitoring Breast Cancer Response to Treatment Using Stokes Shift Spectroscopy of Blood Plasma.

With the emerging trend of personalized cancer treatment, there is a need to develop noninvasive/minimally invasive techniques for treatment monitoring. In this regard, in this work fluorescence analysis of blood plasma of breast cancer patients has been used for the evaluation of response to treatment. This approach delivers information not only about the change in biochemical constituents but also about the altered metabolic pathway. Spectral deconvolution method is employed to compute the fluorescence intensity, peak wavelength, and full-width half maxima for different endogenous fluorophores. The fluorescence measurements were made on blood plasma collected from 10 normal subjects, 10 pre-treated cancer patients, and 10 post-treated patients. Besides, variations in relative concentration of tryptophan, collagen, NADH, and FAD, peak shifts and broadening of peaks are observed for tryptophan, NADH, and FAD, in blood plasma of pre-treated cancer patients indicating both biochemical and microenvironmental changes at cellular level. Further, the spectral profile of blood plasma of post-treated patients found to be similar to blood plasma of normal subjects. Linear discriminant analysis showed that pre-treated and post-treated breast cancer is discriminated with a sensitivity and specificity of 100% and 100% respectively.

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

The serine/threonine protein phosphatase 1 (PP1) inhibitors PPP1R2, PPP1R7, and PPP1R11 are evolutionarily ancient and highly conserved proteins. Four PP1 isoforms, PP1α, PP1ß, PP1γ1, and PP1γ2, exist; three of them except PP1γ2 are ubiquitous. The fact that PP1γ2 isoform is present only in mammalian testis and sperm led to the notion that isoform-specific regulators for PP1γ2 in sperm may be responsible for its function. In this report, we studied these inhibitors, PPP1R2, R7, and R11, to determine their spatial and temporal expression in testis and their regulatory functions in sperm. We show that, similar to PP1γ2, the three inhibitors are expressed at high levels in developing spermatogenic cells. However, the transcripts for the regulators are expressed as unique sizes in testis compared with somatic tissues. The three regulators share localization with PP1γ2 in the head and the principal piece of sperm. We show that the association of inhibitors to PP1γ2 changes during epididymal sperm maturation. In immotile caput epididymal sperm, PPP1R2 and PPP1R7 are not bound to PP1γ2, whereas in motile caudal sperm, all three inhibitors are bound as heterodimers or heterotrimers. In caudal sperm from male mice lacking sAC and glycogen synthase kinase 3, where motility and fertility are impaired, the association of PP1γ2 to the inhibitors resembles immature caput sperm. Changes in the association of the regulators with PP1γ2, due to their phosphorylation, are part of biochemical mechanisms responsible for the development of motility and fertilizing ability of sperm during their passage through the epididymis.

The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility†.

Four isoforms of serine/threonine phosphatase type I, PP1α, PP1ß, PP1γ1, and PP1γ2, are derived from three genes. The PP1γ1 and PP1γ2 isoforms are alternately spliced transcripts of the protein phosphatase 1 catalytic subunit gamma gene (Ppp1cc). While PP1γ1 is ubiquitous in somatic cells, PP1γ2 is expressed exclusively in testicular germ cells and sperm. Ppp1cc knockout male mice (-/-), lacking both PP1γ1 and PP1γ2, are sterile due to impaired sperm morphogenesis. Fertility and normal sperm function can be restored by transgenic expression of PP1γ2 alone in testis of Ppp1cc (-/-) mice. The purpose of this study was to determine whether the PP1γ1 isoform is functionally equivalent to PP1γ2 in supporting spermatogenesis and male fertility. Significant levels of transgenic PP1γ1 expression occurred only when the transgene lacked a 1-kb 3΄UTR region immediately following the stop codon of the PP1γ1 transcript. PP1γ1 was also incorporated into sperm at levels comparable to PP1γ2 in sperm from wild-type mice. Spermatogenesis was restored in mice expressing PP1γ1 in the absence of PP1γ2. However, males from the transgenic rescue lines were subfertile. Sperm from the PP1γ1 rescue mice were unable to fertilize eggs in vitro. Intrasperm localization of PP1γ1 and the association of the protein regulators of the phosphatase were altered in epididymal sperm in transgenic PP1γ1 compared to PP1γ2. Thus, the ubiquitous isoform PP1γ1, not normally expressed in differentiating germ cells, could replace PP1γ2 to support spermatogenesis and spermiation. However, PP1γ2, which is the PP1 isoform in mammalian sperm, has an isoform-specific role in supporting normal sperm function and fertility.

Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals.

In mammals, motility and fertilizing ability of spermatozoa develop during their passage through the epididymis. After ejaculation, sperm undergo capacitation and hyperactivation in the female reproductive tract - a motility transition that is required for sperm penetration of the egg. Both epididymal initiation of sperm motility and hyperactivation are essential for male fertility. Motility initiation in the epididymis and sperm hyperactivation involve changes in metabolism, cAMP (cyclic adenosine mono-phosphate), calcium and pH acting through protein kinases and phosphatases. Despite this knowledge, we still do not understand, in biochemical terms, how sperm acquire motility in the epididymis and how motility is altered in the female reproductive tract. Recent data show that the sperm specific protein phosphatase PP1γ2, glycogen synthase kinase 3 (GSK3), and the calcium regulated phosphatase calcineurin (PP2B), are involved in epididymal sperm maturation. The protein phosphatase PP1γ2 is present only in testis and sperm in mammals. PP1γ2 has a isoform-specific requirement for normal function of mammalian sperm. Sperm PP1γ2 is regulated by three proteins - inhibitor 2, inhibitor 3 and SDS22. Changes in phosphorylation of these three inhibitors and their binding to PP1γ2 are involved in initiation and activation of sperm motility. The inhibitors are phosphorylated by protein kinases, one of which is GSK3. The isoform GSK3α is essential for epididymal sperm maturation and fertility. Calcium levels dramatically decrease during sperm maturation and initiation of motility suggesting that the calcium activated sperm phosphatase (PP2B) activity also decreases. Loss of PP2B results in male infertility due to impaired sperm maturation in the epididymis. Thus the three signaling enzymes PP1γ2, GSK3, and PP2B along with the documented PKA (protein kinase A) have key roles in sperm maturation and hyperactivation. Significantly, all these four signaling enzymes are present as specific isoforms only in placental mammals, a testimony to their essential roles in the unique aspects of sperm function in mammals. These findings should lead to a better biochemical understanding of the basis of male infertility and should lead to novel approaches to a male contraception and managed reproduction.

Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa.

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3ß. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or ß-isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild-type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull-down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function.

Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3ß, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3ß is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3ß, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3ß are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3ß does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

Signaling mechanisms in mammalian sperm motility.

The goal of sperm is to fertilize the oocyte. To achieve that purpose, it must acquire motility in the epididymis and hyperactivated motility in the female reproductive tract. Motility is only achieved when the sperm presents a fully functional flagellum, is capable of producing energy to fuel the movement, and suffers epididymal maturation and capacitation. Since sperm is a transcriptionally silent cell, motility depends on the activation and/or inhibitions of key signaling pathways. This review describes and discusses the main signaling pathways involved in primary and hyperactivated motility, as well as the bioenergetic mechanisms necessary to produce energy to fuel sperm motility. Although the complete human sperm motility process is far from being fully known, we believe that in the upcoming decades extensive progress will be made. Understanding the signaling pathways behind sperm motility can help pinpoint the cause of male infertility and uncover targets for male contraception.

Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility.

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.

Targeted disruption of glycogen synthase kinase 3A (GSK3A) in mice affects sperm motility resulting in male infertility.

The signaling enzyme glycogen synthase kinase 3 (GSK3) exists as two isoforms-GSK3A and GSK3B. Protein phosphorylation by GSK3 has important signaling roles in several cells. In our past work, we found that both isoforms of GSK3 are present in mouse sperm and that catalytic GSK3 activity correlates with motility of sperm from several species. Here, we examined the role of Gsk3a in male fertility using a targeted gene knockout (KO) approach. The mutant mice are viable, but have a male infertility phenotype, while female fertility is unaffected. Testis weights of Gsk3a(-/-) mice are normal and sperm are produced in normal numbers. Although spermatogenesis is apparently unimpaired, sperm motility parameters in vitro are impaired. In addition, the flagellar waveform appears abnormal, characterized by low amplitude of flagellar beat. Sperm ATP levels were lower in Gsk3a(-/-) mice compared to wild-type animals. Protein phosphatase PP1 gamma2 protein levels were unaltered, but its catalytic activity was elevated in KO sperm. Remarkably, tyrosine phosphorylation of hexokinase and capacitation-associated changes in tyrosine phosphorylation of proteins are absent or significantly lower in Gsk3a(-/-) sperm. The GSK3B isoform was present and unaltered in testis and sperm of Gsk3a(-/-) mice, showing the inability of GSK3B to substitute for GSK3A in this context. Our studies show that sperm GSK3A is essential for male fertility. In addition, the GSK3A isoform, with its highly conserved glycine-rich N terminus in mammals, may have an isoform-specific role in its requirement for normal sperm motility and fertility.

Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice.

The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (-/-) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa.

BACKGROUND: Protein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa. RESULTS: Here we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures. CONCLUSIONS: The conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract.

Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.

The fibrous sheath (FS) is a flagellar cytoskeletal structure unique to sperm that surrounds the outer dense fibers and axoneme. Its primary components are A-kinase anchoring proteins (AKAPs) 3 and 4, which suggests that the FS affects flagellar beating via the scaffolding of signaling pathways necessary for motility. Sperm proteins ROPN1 and ROPN1L bind AKAP3. To determine the role of ROPN1 and ROPN1L in sperm function, we created mice deficient in ROPN1 (RKO), mice deficient in ROPN1L (RLKO), and double knockout mice (DKO). All three strains of mice had normal testicular morphology and spermatogenesis. Only the DKOs had obvious defects in sperm morphology (thinning and shredding of the principal piece), which was accompanied by a reduction in AKAP3 levels. RLKO mice had slightly reduced sperm motility and increased levels of ROPN1. RKO mice had moderately impaired motility and increased levels of ROPN1L. DKO sperm were immotile. We have previously determined that RKO male mice are subfertile, and DKO males are infertile. Together these data indicate that ROPN1L and ROPN1 compensate for each other in the absence of the opposing protein, possibly to maintain AKAP3 incorporation in the FS. Sperm from mice lacking ROPN1L exhibited reductions in both cAMP-dependent protein kinase (PKA) phosphorylation of a 270-kDa protein (perhaps FSCB), and in capacitation-induced tyrosine phosphorylation. Sperm from mice lacking ROPN1 had reduced levels of FSCB and increased tyrosine phosphorylation of noncapacitated sperm. These data demonstrate that mutations in ROPN1 and ROPN1L can cause defects in FS integrity, sperm motility, and PKA-dependent signaling processes, leading to male infertility.

Significant expression levels of transgenic PPP1CC2 in testis and sperm are required to overcome the male infertility phenotype of Ppp1cc null mice.

PPP1CC2, one of four isoforms of the ser/thr protein phosphatase PP1, is a mammalian-specific splice variant of the Ppp1cc gene, and the only isoform whose expression is confined almost completely to spermatogenic cells. Additionally, PPP1CC2 is the sole isoform found in mammalian spermatozoa. Although PPP1CC1, the other Ppp1cc product, is expressed in many tissues including testis, the only phenotype resulting from deletion of Ppp1cc gene is male infertility. To determine which of the products of Ppp1cc is essential for male fertility, we created two PPP1CC2 transgenes, eTg-G2 and pTg-G2, where Ppp1cc2 expression was driven by the putative endogenous promoter of Ppp1cc or by the testis specific human Pgk2 promoter, respectively. Our results demonstrate that the 2.6-kb genomic region directly upstream of the Ppp1cc structural gene can drive expression of Ppp1cc2, and recapitulate the wild-type tissue specificity of PPP1CC2 in transgenic mice. More importantly, we show that expression of PPP1CC2 alone, via either promoter, is able not only to restore normal spermatogenesis, but the fertility of Ppp1cc null mice as well, provided that transgenic PPP1CC2 expression in testis reaches at least a lower threshold level equivalent to approximately 50% of its expression by a Ppp1cc +/- male. We conclude that the endogenous Ppp1cc promoter normally functions in the testis to maintain a sufficient level of PPP1CC2 expression for normal spermatogenesis to occur, and that production of spermatozoa capable of fertilization in vivo can take place in the complete absence of PPP1CC1 expression.

Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development.

BACKGROUND: The 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (ß, γ, ε, ζ, η, τ and, σ). These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. RESULTS: We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (ß, ε, γ, and ζ) are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. CONCLUSIONS: We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the differential expression of these 14-3-3 isoforms in female germ cells and ovarian follicles provides the foundation for further investigating 14-3-3 isoform-specific interactions with key proteins involved in ovarian development, meiosis and oocyte maturation. This will lead to a better understanding of the individual functional roles of the 14-3-3 protein isoforms in mammalian oogenesis and female reproductive development.

Identification of testis 14-3-3 binding proteins by tandem affinity purification.

The 14-3-3 family of proteins interacts with various cellular phosphoproteins and regulates multiple cell signaling cascades. Identification of 14-3-3 interactors is important to define 14-3-3 functions in various biological pathways. The binding partners of protein 14-3-3 in testis are not known. The main goal of this study was to identify the 14-3-3 interactome in testis to determine the 14-3-3 regulated cellular processes in testis. We used transgenic mice expressing tandem affinity tagged 14-3-3ζ (TAP-14-3-3ζ) driven by the ubiquitin promoter to isolate 14-3-3 binding proteins. The 14-3-3 complexes in testis were isolated using a two-step tandem affinity purification (TAP) followed by identification with liquid chromatography/tandem mass spectrometry (LC-MS/MS). A total of 135 proteins were found to be associated with 14-3-3 in vivo in testis. Comparison of the testis 14-3-3 proteome with known 14-3-3 binding proteins showed that 71 of the proteins identified in this study are novel 14-3-3 interactors. Eight of these novel 14-3-3 interacting proteins are predominantly expressed in testis. The 14-3-3 interactors predominant in testis are: protein phosphatase1γ2 (PP1γ2), spermatogenesis associated 18 (SPATA18), phosphoglycerate kinase-2 (PGK2), testis specific gene A-2 (TSGA-2), dead box polypeptide 4 (DDX4), piwi homolog 1, protein kinase NYD-SP25 and EAN57. The fact that some of these proteins are indispensable for spermatogenesis suggests that their binding to 14-3-3 may be important for their function in germ cell division and maturation. These findings are discussed in context of the putative functions of 14-3-3 in spermatogenesis.