Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Stimulation by thyrotropin, long-acting thyroid stimulator, and dibutyryl 3′, 5′-adenosine monophosphate of protein and ribonucleic acid synthesis and ribonucleic acid polymerase activities in porcine thyroid in Vitro

The in vitro incorporation of [3H]uridine into RNA and [3H]leucine into protein in slices of porcine thyroid was studied. Thyrotropin (10-500 mU/ml of medium), when added with [3H]uridine, inhibited incorporation into RNA, but as little as 10 mU of thyrotropin per ml stimulated incorporation of [3H]orotic acid into RNA. Uridine kinase (EC 2.7.1.48) was found to be inhibited in slices incubated with thyrotropin whereas UMP 5′ nucleotidase (EC 2.1.3.5) was not. Preincubation of slices with thyrotropin (5-50 mU/ml) led to enhanced incorporation of subsequently added [3H]uridine and [3H]leucine. When slices were preincubated with long-acting thyroid stimulator-IgG (2.5 or 5 mg per ml of medium) incorporation of [3H]uridine and [3H]leucine was similarly enhanced, with the smaller concentration being more effective. Without preincubation these stimulatory effects were mimicked by 1 mM dibutyryl 3′,5′-AMP and, to a lesser extent, 1 mM 3′,5′-AMP. AMP and ATP also stimulated [3H]uridine incorporation in this system but only after more prolonged periods of incubation than were required for the other nucleotides. RNA polymerase (EC 2.7.7.6) activity measured in isolated thyroid nuclei had two components, one Mg2+-stimulated and the other requ ring Mn2+ and high salt content [0.4 M (NH4)2SO4]. These activities, and particularly the former, were enhanced if thyroid slices were incubated with thyrotropin (5-100 mU/ml of medium), 2.5 mg or 5.0 mg of long-acting thyroid stimulator-IgG per ml, or 1 mM dibutyryl 3′,5′-AMP, before isolatior of the nuclei and measurement of enzyme activities; 1 mM AMP, ADP, or 2′,3′-GMP had no influence. Added directly to the nuclei, thyrotropin, long-acting thyroid stimulator-IgG, and dibutyryl 3′,5′-AMP had no effect on RNA polymerase activities. These data are seen as affording evidence for mediation by 3′,5′-AMP of effects of thyrotropin and long-acting thyroid stimulator on thyroid RNA and protein synthesis, at least in part through an indirect stimulation of nuclear RNA polymerase activities.

Isolation and characterization of a gonadotropin receptor binding inhibitor from porcine follicular fluid

The presence of a gonadotropin receptor binding inhibitor in pooled porcine follicular fluid has been demonstrated. Porcine follicular fluid fractionation on DE-32 at near neutral pH, followed by a cation exchange chromatography on SPC-50 and Cibacron blue affinity chromatography, yielded a partially purified gonadotropin receptor binding inhibitor (GI-4). The partially purified GI binding inhibitor inhibited the binding of both 125I labelled hFSH and hCG to rat ovarian receptor preparation. SDS electrophoresis of radioiodinated partially purified GI followed by autoradiography made it possible to identify the binding component as a protein of molecular weight of 80000. Subjecting 125I labelled GI-4 to chromatography on Sephadex G-100 helped obtain a homogeneous material, Gl-5. The 125I labelled GI-5 exhibited in its binding to ovarian membrane preparations characteristics typical of a ligand-receptor interaction such as saturability, sensitivity to reaction conditions as time, ligand and receptor concentrations and finally displaceability by unlabelled inhibitor as well as FSH and hCG in a dose dependent manner. This material could bind ovarian receptors for both FSH and LH, its binding being inhibited by added FSH or hCG in a dose dependent manner.

Effect of 80 kDa protein of porcine follicular fluid on gonadotropin stimulated progesterone production in rat granulosa cells in vitro

We reported the presence of a 80 kDa polypeptide in porcine follicular fluid that inhibited the binding of 125I-radiolabelled hFSH as well as hCG to the rat ovarian gonadotropin receptors. In the present study, the biological activity of the receptor binding inhibitor is determined using an in vitro bioassay procedure. Granulosa cells isolated from PMSG primed immature rat ovaries respond to exogenously added gonadotropins in terms of progesterone production. Addition of fractions containing the gonadotropin receptor binding inhibitory activity inhibited progesterone production stimulated by the gonadotropins in a dose-dependent fashion. The receptor binding inhibitory activity was also capable of inhibiting progesterone production stimulated by PMSG, which has both FSH- and LH-like activities in rats. In contrast, progesterone production stimulated by dbcAMP was not inhibited by the receptor binding inhibitor. This result indicates that the site of action of the inhibitor is proximal to the formation of the cAMP. The above observations point out to a possible role for this factor in modulating gonadotropin activity at the ovarian level.

Pentoxifylline improves sperm capacitation and in vitro fertilization of oocytes in the golden hamster

Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri- implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 5] motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and greater than or equal to 1.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 +/- 7.8) than for the controls (69.5 +/- 10.2), whereas, treatment with 0.45 mM PF increased the rates (91.6 +/- 4.3) compared with that of the controls (80.2 +/- 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster.

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+](i)) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline [0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoal, cGMP values (similar to 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/HCO(3)(-)antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 mu M) A protein kinase G inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR, A phospholipase A(2) inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+](i) was measured using fura-2-AM, there was an early rise 271 nM at 0.5 hi in pentoxifylline(-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR(20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-](i) and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A;A(2) and protein kinase C activity.

Effects of elastase and collagenase on the nonlinearity and anisotropy of porcine aorta

We use enzymatic manipulation methods to investigate the individual and combined roles of elastin and collagen on arterial mechanics. Porcine aortic tissues were treated for differing amounts of time using enzymes elastase and collagenase to cause degradation in substrate proteins elastin and collagen and obtain variable tissue architecture. We use equibiaxial mechanical tests to quantify the material properties of control and enzyme treated tissues and histological methods to visualize the underlying tissue microstructure in arterial tissues. Our results show that collagenase treated tissues were more compliant in the longitudinal direction as compared to control tissues. Collagenase treatment also caused a decrease in the tissue nonlinearity as compared to the control samples in the study. A one hour collagenase treatment was sufficient to cause fragmentation and degradation of the adventitial collagen. In contrast, elastase treatment leads to significantly stiffer tissue response associated with fragmented and incomplete elastin networks in the tissue. Thus, elastin in arterial walls distributes tensile stresses whereas collagen serves to reinforce the vessel wall in the circumferential direction and also contributes to tissue anisotropy. A microstructurally motivated strain energy function based on circumferentially oriented medial fibers and helically oriented collagen fibers in the adventitia is useful in describing these experimental results.

Structural Transformations in Protein Crystals caused by controlled Dehydration

Recent experiments in this laboratory on structural transformations caused by controlled dehydration of protein crystals have been reviewed. X-ray diffraction patterns of the following crystals have been examined under varying conditions of environmental humidity in the relative humidity range of 100-75%: a new crystal form of bovine pancreatic ribonuclease A grown from acetone solution in tris buffer (I), the well-known monoclinic form of the protein grown from aqueous ethanol (II), the same form grown from a solution of 2-methyl pentan-2,4-diol in phosphate buffer (III), tetragonal (IV), orthorhombic (V), monoclinic (VI) and triclinic (VII) hen egg white lysozyme, porcine 2 Zn insulin (VIII), porcine 4 Zn insulin (IX) and the crystals of concanavalin A(X). I, II, IV, V and VI undergo one or more transformations as evidenced by discontinuous changes in the unit cell dimensions, the diffraction pattern and the solvent content. Such water-mediated transformations do not appear to occur in the remaining crystals in the relative humidity range explored. The relative humidity at which the transformation occurs is reduced when 2-methyl pentan-2,4-diol is present in the mother liquor. The transformations are affected by the crystal structure but not by the amount of solvent in the crystals. The X-ray investigations reviewed here and other related investigations emphasize the probable importance of water-mediated transformations in exploring hydration of proteins and conformational transitions in them.

Pentoxifylline induces hyperactivation and acrosome reaction in spermatozoa of golden hamsters: changes in motility kinematics

Pentoxifylline (PF) is used to enhance motility of spermatozoa from infertile human subjects. We have previously shown that 0.45 mM PF improved capacitation of spermatozoa and fertilization of oocytes in vitro in hamsters. The present study was carried out to assess PF- induced changes in motility kinematics of hamster spermatozoa by a computer-aided sperm analyser (CASA) and determine the timing of onset of hyperactivation (HA) and acrosome reaction (AR) in PF-treated spermatozoa. Motility kinematics were analysed by CASA for 0-8 h in the absence or presence of 0.45 mM PF in Tyrode's medium supplemented with lactate, pyruvate and polyvinyl alcohol (TLP-PVA) or in TLP-PVA with bovine serum albumin (TALP-PVA). Conventional assessment was also made on the percentage of motility and quality of motility of spermatozoa; values were expressed as sperm motility index (SMI). Both in TALP-PVA and TLP-PVA, PF markedly increased SMI, especially the quality of motility (P < 0.02) by 2-3 h which was sustained up to 6 h. The motility kinematic data of PF-treated spermatozoa in TALP-PVA showed that average path velocity, curvilinear velocity and amplitude of lateral head displacement significantly (P < 0.05) increased as early as 2 h, with the expected decrease in straightness (STR) and linearity (LIN). Similar changes were also observed with PF-treated spermatozoa in TLP-PVA. Moreover, the percentage of hyperactivated spermatozoa in PF-treated samples was significantly (P < 0.001) higher than the untreated control at 2 h. To determine whether PF could induce AR, independent of bovine serum albumin, quantitative AR was assessed by observing the presence or absence of acrosomal cap on viable spermatozoa. PF significantly (P < 0.001) increased the percentage of AR as early as 2 h, reaching maximum at 4 h both in TALP-PVA (P < 0.05) and in TLP-PVA (P < 0.001). These results show that, in hamsters, PF induces early onset (by 2 h) of HA and AR and increases the proportion of spermatozoa undergoing physiological maturation.

Enzymatic Degradation of Poly(soybean oil-g-methyl methacrylate)

This study discusses grafting of methyl methacrylate units from thepolymeric soybean oil peroxide to produce poly(soybean oil-graft-methyl methacrylate) (PSO-g-PMMA). The degradation of this copolymer in solution was evaluated in the presence of different lipases, viz Candida rugosa (CR), Lipolase 100T (LP), Novozym 435 (N435) and Porcine pancreas (PP), at different temperatures The copolymer degraded by specific chain end scission and the mass fraction of the specific product evolved was determined The degradation was modeled using continuous distribution kinetics to determine the rate coefficients ofmenzymatic chain end scission and deactivation of the enzyme The enzymes, CR. LP and N435 exhibited maximum activity for the degradation of PSO-g-PMMA at 60 degrees C, while PP was most active at 50 degrees C. The thermal degradability of the copolymer, assessed by thermo-gravimetry, indicated that the activation energy of degradation of the copolymer was 154 kJ mol(-1), which was lesser than that of the PMMA homopolymer.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.