Joseph S. Tash

Flagellar Motility Requires the CAMP-Dependent Phosphorylation of a Heat-Stable NP-40-Soluble 56 kd Protein, Axokinin

Using NP-40-treated dog sperm as a model, the stimulatory effect of cAMP upon reactivated flagellar motility has been shown to be dependent upon the cAMP-dependent phosphorylation of a heat-stable NP-40-soluble protein of 56 kd. Examination by two-dimensional polyacrylamide gel electrophoresis of NP-40 extract proteins phosphorylated with gamma-32P-ATP revealed a major cAMP-dependent phosphopeptide at 56 kd. This is the only cAMP-dependent phosphoprotein common to NP-40 extracts of all tissues that show cAMP-dependent stimulation of flagellar motility. These cells and tissues include sea urchin, dog, and human sperm, as well as dog trachea and retina. Moreover, this phosphoprotein is absent in nonstimulatory extracts from tissues such as skeletal muscle, brain, and liver. We conclude that the cAMP-dependent phosphorylation of the 56 kd peptide represents a major regulatory component of not only sperm but other types of axonemal motility as well.

Phosphorylation of hen progesterone receptor by cAMP dependent protein kinase.

Abstract Progesterone receptor A and B subunits from laying hen oviducts were highly purified and their phosphorylation by cAMP-dependent protein kinase from bovine heart was studied. Both proteins are phosphorylated by the kinase using physiological or subphysiological concentrations of the enzyme. This result indicates that the receptors are good substrates. The reaction is dependent upon exogenous enzyme; no phosphorylation is seen in the absence of protein kinase.

Role of FYN Kinase in Spermatogenesis: Defects Characteristic of Fyn-Null Sperm in Mice

ABSTRACT FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.

Regulation of Human Sperm Motility and Hyperactivation Components by Calcium, Calmodulin, and Protein Phosphatases

The role of Ca2+, calmodulin, and protein phosphatases on motility and hyperactivation of noncapacitated, capacitating, and detergent-permeabilized reactivated human sperm was examined. In noncapacitated sperm, W7 inhibited percent motility (%MOT), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and percent hyperactivation (%HYP) in an extracellular Ca2+ concentration-dependent manner (p < .05). However, in capacitating sperm, inhibition of motility by W7 was independent of external Ca2+. Treatment of reactivated sperm with a synthetic calmodulin inhibitor peptide decreased VCL and ALH in a Ca(2+)-dependent manner (p < .05). Ca2+ exhibited a dramatic influence on motility within a narrow concentration range (0.7 to 1.0 microM) in reactivated sperm. A calmodulin-dependent protein phosphatase (PP2B) was identified by activity assay, immunoblotting, and dephosphorylation of endogenous phosphoproteins. The sperm enzyme, unlike bovine brain PP2B, was inhibited by 1 microM okadaic acid (OA) in the presence of Mn2+, suggesting that the sperm enzyme is unique. In reactivated sperm, inhibition of endogenous PP2B-like activity with anti-PP2B antibodies altered ALH, whereas OA altered both VCL and ALH and also inhibited a subset of Ca(2+)-dependent dephosphorylations of cAMP-dependent phosphoproteins in capacitating sperm. These results suggest (1) an important role for calmodulin and PP2B in Ca(2+)-regulated motility parameters, particularly ALH, and (2) that modulation of human sperm motility, including hyperactivation by cAMP-dependent phosphorylation, requires calmodulin-dependent as well as other protein dephosphorylations.

Gamendazole, an Orally Active Indazole Carboxylic Acid Male Contraceptive Agent, Targets HSP90AB1 (HSP90BETA) and EEF1A1 (eEF1A), and Stimulates Il1a Transcription in Rat Sertoli Cells

Gamendazole was recently identified as an orally active antispermatogenic compound with antifertility effects. The cellular mechanism(s) through which these effects occur and the molecular target(s) of gamendazole action are currently unknown. Gamendazole was recently designed as a potent orally active antispermatogenic male contraceptive agent. Here, we report the identification of binding targets and propose a testable mechanism of action for this antispermatogenic agent. Both HSP90AB1 (previously known as HSP90beta [heat shock 90-kDa protein 1, beta]) and EEF1A1 (previously known as eEF1A [eukaryotic translation elongation factor 1 alpha 1]) were identified as binding targets by biotinylated gamendazole (BT-GMZ) affinity purification from testis, Sertoli cells, and ID8 ovarian cancer cells; identification was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and Western blot analysis. BT-GMZ bound to purified yeast HSP82 (homologue to mammalian HSP90AB1) and EEF1A1, but not to TEF3 or HBS1, and was competed by unlabeled gamendazole. However, gamendazole did not inhibit nucleotide binding by EEF1A1. Gamendazole binding to purified Saccharomyces cerevisiae HSP82 inhibited luciferase refolding and was not competed by the HSP90 drugs geldanamycin or novobiocin analogue, KU-1. Gamendazole elicited degradation of the HSP90-dependent client proteins AKT1 and ERBB2 and had an antiproliferative effect in MCF-7 cells without inducing HSP90. These data suggest that gamendazole may represent a new class of selective HSP90AB1 and EEF1A1 inhibitors. Testis gene microarray analysis from gamendazole-treated rats showed a marked, rapid increase in three interleukin 1 genes and Nfkbia (NF-kappaB inhibitor alpha) 4 h after oral administration. A spike in II1a transcription was confirmed by RT-PCR in primary Sertoli cells 60 min after exposure to 100 nM gamendazole, demonstrating that Sertoli cells are a target. AKT1, NFKB, and interleukin 1 are known regulators of the Sertoli cell-spermatid junctional complexes. A current model for gamendazole action posits that this pathway links interaction with HSP90AB1 and EEF1A1 to the loss of spermatids and resulting infertility.

Prolactin-like protein-A is a functional modulator of natural killer cells at the maternal-fetal interface.

Natural killer (NK) cells are the predominant lymphocytes present in healthy rodent and human implantation sites. In the rat, the expansion, differentiation and subsequent migration of NK cells away from the developing chorioallantoic placenta coincide with the expression of a novel pregnancy- and trophoblast cell-specific cytokine, prolactin (PRL)-like protein A (PLP-A). PLP-A specifically binds to uterine NK cells but does not appear to utilize receptor systems for PRL. In the present report, we show that PLP-A interactions with NK cells are not mediated by receptors utilized by known modulators of NK cell function, including interleukin-2, interleukin-7, interleukin-12, and interleukin-15 (IL-15). Uterine NK cells respond to PLP-A or IL-15 with an increase in intracellular calcium mobilization. In contrast, PLP-A, unlike IL-15, effectively suppresses the ability of NK cells to produce interferon-gamma (IFNgamma), a key mediator of NK cell function. Placental PLP-A expression is reciprocal to mesometrial decidua expression of IFNgamma. Increased expression of PLP-A by the placenta coincides with the decline of IFNgamma content in the mesometrial decidua adjacent to the placenta. In summary, trophoblast cell-derived PLP-A contributes to the regulation of NK cells at the maternal-fetal interface to ensure appropriate embryonic growth and development.

A Novel Potent Indazole Carboxylic Acid Derivative Blocks Spermatogenesis and Is Contraceptive in Rats after a Single Oral Dose

Women have historically been the focus for development of new contraceptive methods. The National Institutes of Health, World Health Organization, and Institute of Medicine have stressed the need to develop nonhormonal, nonsteroidal male contraceptive agents. We report results from initial dose-ranging studies of a new indazole carboxylic acid analogue, gamendazole. An infertility rate of 100% was achieved in seven out of seven proven-fertile male rats 3 wk after a single oral dose of 6 mg/kg of gamendazole. Fertility returned by 9 wk in four of seven animals, with typical numbers of normal-appearing conceptuses. A fertility rate of 100% returned in four of six animals that became infertile at a single oral dose of 3 mg/kg of gamendazole. No differences in mating behavior were observed in either of the gamendazole-treated groups versus the control (vehicle-only) group. In the animals that showed reversible infertility, a transient increase in circulating FSH levels coincided with an initial decline in inhibin B levels after administration of gamendazole, but no other significant changes in circulating reproductive hormones were observed. Gamendazole inhibited production of inhibin B by primary Sertoli cells in vitro with a median inhibitory concentration of 6.8 thorn+/- 3.0 (SEM) (3/4)x 10(-10) M, suggesting that Sertoli cells are a primary target. A biotinylated gamendazole analogue revealed cytoplasmic and perinuclear binding of gamendazole in primary Sertoli cells. Gamendazole represents the most potent new oral antispermatogenic indazole carboxylic acid to date. Our results, however, demonstrate that additional dose-finding studies are required to improve reversibility and widen the therapeutic window before more detailed drug development of this potential nonhormonal male contraceptive agent can occur.

[59] Ca2+ Regulation of sperm axonemal motility

Publisher Summary Calcium and cAMP are two pivotal intracellular regulators of sperm flagellar motility. cAMP is required for the initiation and maintenance of axonemal motility. Dog spermatozoa are used to investigate the role of cAMP and calcium in the regulatory mechanisms of axonemal motility. Washed dog sperm were permeabilized with detergent and reactivated with ATP in the presence of cAMP and/or calcium. Once the details of model preparation had been worked out, ATP was replaced by [γ- 32 P] ATP under identical experimental conditions to ask the question whether protein phosphorylation might be involved. Using this model system, specific peptides are identified, which are pivotal regulators of flagellar motility not only in dog sperm, but in other axonemal systems as well. With regard to the action of cAMP, a single cAMP-dependent phosphoprotein is identified whose phosphorylation is obligatory for initiation and maintenance of flagellar motility. This peptide, named axokinin, is ubiquitous to all axonemal sources examined and is not only required, but is sufficient to induce flagellar reactivation provided it has been phosphorylated. Using the same techniques, specific proteins involved in the regulation of axonemal motility by calcium are recently identified. Furthermore, the effects of calcium on motility and protein phosphorylation appear to be modulated by calmodulin and specific proteins with which the calmodulin interacts in a calcium-dependent manner.

Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases.

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009-0.0015 μM) from a single hit compound with weak inhibitory activity (IC50 = 15 μM), discovered by high-throughput screening. Structure-based design was performed using 35 cocrystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4, and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 μM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics.

Synthesis and Evaluation of Eight- and Four-Membered Iminosugar Analogues as Inhibitors of Testicular Ceramide-Specific Glucosyltransferase, Testicular β-Glucosidase 2, and Other Glycosidases

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve ring-closing metathesis and Sharpless asymmetric dihydroxylation and for the four-membered analogues Sharpless epoxidation, epoxide ring-opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6) was moderately active against rat-derived ceramide-specific glucosyltransferase, and four of the other eight-membered analogues were weakly active against rat-derived β-glucosidase 2. Among the four-membered analogues, ((2R,3S,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25) displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of β-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived β-glucosidase, and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC(50) of 600 nM and β-glucosidase (almond) with an IC(50) of 20 μM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied.