Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea

The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.     

A cAMP-binding phosphoprotein in Drosophila heads is similar to the regulatory subunit of the mammalian type II cAMP-dependent protein kinase

Cyclic AMP (cAMP)-dependent regulation of in vitro phosphorylation of several proteins including a cAMP-binding protein was studied with crude membrane and cytosol fractions from Drosophila heads. Phosphorylation of at least seven distinct proteins was enhanced in the presence of cAMP. Interestingly, however, the phosphorylation of a 56 kDa protein was apparently reduced by cAMP in the membrane but not in the cytosol fraction. The following data strongly indicate that the 56 kDa phosphoprotein in both membrane and cytosol fractions is a cAMP-binding protein, very similar to the regulatory subunit (RII) of a mammalian cAMP-dependent protein kinase, and that its binding to cAMP makes this protein very susceptible to the action of phosphatases: (i) cAMP highly stimulated the dephosphorylation of the 56 kDa phosphoprotein by the endogenous phosphatase in the membrane fraction. (ii) The dephosphorylation of a similar 56 kDa phosphoprotein in the cytosol fraction by an exogenous, cAMP-independent, alkaline phosphatase was also highly stimulated by cAMP. (iii) The 56 kDa phosphoprotein was covalently bound to cAMP by u.v. irradiation. (iv) The alkaline-phosphatase treatment reversibly converted this phosphoprotein to a 53 kDa non-phosphorylated protein. (v) The 53 kDa protein was selectively bound to cAMP-agarose and subsequently eluted by cAMP and high salt. (vi) This protein served as a substrate for the catalytic subunit of a mammalian cAMP-dependent protein kinase.     

Neuronal microtubule-associated protein 2D is a dual a-kinase anchoring protein expressed in rat ovarian granulosa cells

A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype.     

Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. cDNA and deduced amino acid sequence of rat P-450scc

We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Expression of CCAAT/enhancer binding proteins alpha and beta in the porcine ovary and regulation in primary cultures of granulosa cells

CCAAT/enhancer binding proteins alpha and beta (CEBPA/ CEBPB) were evaluated in the porcine ovary during the estrous cycle. CEBPB mRNA was present in antral follicles and was significantly increased in healthy corpora lutea (CL), whereas CEBPA mRNA was constitutively expressed in these structures. Both isoforms of CEBPA (42 and 30 kDa) exhibited greater expression in preovulatory follicles, and the 42-kDa isoform increased in CL, whereas the 30-kDa isoform decreased. All major isoforms of CEBPB (38, 34, and 20 kDa) were expressed, with the 34- and 20-kDa isoforms being more abundant in preovulatory follicles and further increased in CL. The effects of FSH and cAMP analogue on the distribution of CEBP isoforms were evaluated in primary cultures of porcine granulosa cells. FSH and 8-Br-cAMP had little stimulatory effect on isoform distribution, but cAMP treatment for 24 h tended to decrease the 30-kDa form of CEBPA and the 34-kDa form of CEBPB. The 34-kDa form of CEBPB was decreased by the protein kinase A inhibitor H89 at 4 h (with FSH treatment), and by both protein kinase A and phosphatidylinositol 3-kinase inhibitors at 24 h of treatment. In transfected granulosa cells, FSH and cAMP analogue stimulated a CEBP consensus sequence-reporter construct that was blocked by H89. These data implicate protein kinase A as the major regulator of CEBPB isoform distribution and CEBP-mediated transactivation in granulosa cells. The differential expression of specific CEBPA/B isoforms observed in maturing follicles and CL may contribute to changes in follicular cell differentiation and increasing steroidogenic capacity.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Inhibitory effect of the regulatory subunit of type I cAMP-dependent protein kinase on phosphoprotein phosphatase

The regulatory subunit of type I cAMP-dependent protein kinase (RI) from rabbit skeletal muscle inhibited the activity of a low molecular weight phosphoprotein phosphatase. The inhibition was concentration and time dependent. A maximum inhibition, about 70%, was observed at 2 microM of RI with an apparent Ki of 0.8 microM. Inhibition was associated with a decrease in Vmax with no change in Km for substrate, phosphorylase a. On the other hand, cAMP-dependent protein kinase holoenzyme or its catalytic subunit was without any effect. The inhibition of phosphoprotein phosphatase by RI may be of physiological significance since the dissociation of cAMP-dependent protein kinase by cAMP would result in a simultaneous increase in the phosphorylation and decrease in the dephosphorylation rates of target proteins.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets

Summary Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvae kinase indicate it is the M₂ isoenzyme and that its phosphorylation does not affect its catalytic activity.     

Cyclic AMP increasing agents rapidly stimulate vimentin phosphorylation in quiescent cultures of Swiss 3T3 cells

The results presented here demonstrate that an elevation in the cellular levels of cyclic AMP (cAMP) increases the phosphorylation of an Mr = 58,000 cellular protein in quiescent cultures of Swiss 3T3 cells. The enhancement of 32Pi incorporation into the Mr 58,000 cellular protein was detected as early as 1 min and reached a maximum after 20 min of treatment. The role of cAMP in the phosphorylation of Mr = 58,000 protein is substantiated by the following lines of evidence: a) a variety of agents that cause cAMP accumulation in 3T3 cells, including cholera toxin, 5'-N-ethylcarboxamideadenosine (NECA), PGE1, and 3-isobutyl-1-methyl-xanthine (IBMX) increased the phosphorylation of the same Mr 58,000 cellular protein as demonstrated by peptide mapping; b) inhibitors of cyclic nucleotide phosphodiesterase potentiated the ability of low concentrations of the adenylate cyclase activators NECA, PGE1, and forskolin to increase Mr 58,000 phosphorylation; and c) permeable derivatives of cAMP such as 8BrcAMP were also effective and specific in promoting Mr 58,000 phosphorylation. Detergent extraction, immunoblotting, and immunoprecipitation identified the Mr = 58,000 phosphoprotein as vimentin, the main protein subunit of the intermediate filaments of mesenchymal cells including Swiss 3T3 cells. Studies with intact 3T3 cells revealed that an increase in the intracellular level of cAMP induced a marked redistribution and collapse of the intermediate filaments. These results raise the possibility that an intact intermediate filament network may restrict the reinitiation of DNA synthesis.     

Hormonal regulation of nuclear cyclic AMP-dependent protein kinase subunit levels in rat ovaries

Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.     

Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.

The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.