Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 mu l of fertilization medium (FM); (ii) 250 mu l of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 mu l of FM with 250 mu l of NLAUTF and 4 mu l of RCA-1 lectin; (iv) 250 mu l of FM with 250 mu l of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 mu l of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 mu g heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Influence of irradiant energy on degree of conversion, polymerization rate and shrinkage stress in an experimental resin composite system

Objective. This study evaluated the degree of conversion (DC), maximum rate of cure (R(p)(max)), and polymerization stress (PS) developed by an experimental dental composite subjected to different irradiant energies (3,6,12, 24, or 48J/cm(2)) under constant irradiance (500 mw/cm(2)). Methods. DC and R(p)(max) were monitored for 10 min on the bottom surface of 2-mm thick disks and on 150-mu m thick films (representing the top of the specimen) using ATR-FTIR. PS was monitored for 10 min in 2-mm thick disks bonded to two glass rods (O = 5 mm) attached to a universal testing machine. One-way ANOVA/Tukey tests were used and differences in DC and R(p)(max) between top and bottom surfaces were examined using Student`s t-test. Statistical testing was performed at a pre-set alpha of 0.05. Results. For a given surface, DC showed differences among all groups, except at the top between 24 and 48 J/cm(2). R(p)(max) was similar among all groups at the same surface and statistically higher at the top surface. PS also showed significant differences among all groups. Data for 48 J/cm(2) were not obtained due to specimen failure at the glass/composite interface. Significance. Increases in irradiant exposure led to significant increases in DC and PS, but had no effect on R(p)(max) (c) 2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Mice with genetic deletion of the heparin-binding growth factor midkine exhibit early preclinical features of Parkinson`s disease

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson`s disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.