Josine Beek

Differential apoptotic staining of mammalian blastocysts based on double immunofluorescent CDX2 and active caspase-3 staining

Several approaches have been described for differential staining of blastocysts, but these methods are often time-consuming and unreliable. Here we describe a method for simultaneous differential staining and detection of apoptosis. The differential staining is based on the transcription factor CDX2 which is localized in the nucleus of trophectoderm (TE) cells but absent in the inner cell mass (ICM). Apoptosis is detected by staining of active caspase-3, a key player in several apoptotic pathways. This new approach represents a robust method for quantifying simultaneously ICM/TE ratio and apoptotic cell ratio in bovine, murine, porcine, and human blastocysts.

Boar seminal plasma components and their relation with semen quality

Select boar seminal plasma (SP) components and their relation to semen quality were investigated. Thirty nine boars from three artificial insemination (AI) centers were divided into group A (GA: > 80% normal sperm and >70% motility) and group B (GB: < 80% normal sperm and < 70% motility). Each ejaculate was collected and semen volume, concentration, sperm motility (computer aided semen analysis; CASA), morphology, and vitality (both eosin nigrosin staining) were investigated. The SP was separated and analyzed for aspartate-amino-transferase (AST), γ-glutamyl-transferase (GGT), alkaline phosphatase (ALP) activity, and the concentrations of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphate (PO43-), magnesium (Mg), selenium (Se) and zinc (Zn) were assessed. Repeated measures (2 months interval) were conducted in eight boars of GA from one AI center. The activity of GGT (r = -0.482) and ALP (r = -0.459) was moderately associated (p < 0.05) with ejaculate volume and strongly associated with concentration (r = 0.580 and r = 0.618, respectively; p = 0.000). Moderate associations (p < 0.05) were found between ALP (r = 0.439), GGT (r = 0.387), Na (r = -0.428), K (r = 0.354), and Se (r = 0.354) with progressive motility. The SP concentration of Na (r = -0.401), Cl (r = -0.521), and K (r = 0.350) was associated (p < 0.05) with normal morphology. Only Mg was associated (p < 0.05) with membrane damage (r = -0.335). The concentration of Na, Cl, and Zn (1681.0 vs. 1701.0 µg/dL) was different between groups (p < 0.05). Repeated measures showed significant differences in time but only for Na, Mg, and Zn (p < 0.05). In conclusion, several biochemical components of SP were related to semen quality. The analysis of biochemical parameters could provide extra information about reproductive health of AI boars.

Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro

In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1-TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.

A critical assessment of the effect of serine protease inhibitors on porcine fertilization and quality parameters of porcine spermatozoa in vitro

Proteases play an important role during mammalian fertilization. Their function is frequently investigated using specific inhibitors. We analyzed four serine protease inhibitors [4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor from glycine max (STI), Nα-tosyl-L-lysine-chloromethyl ketone hydrochloride (TLCK) and N(p)-tosyl-L-phenylalanine-chloromethyl ketone (TPCK)] for their in vitro effect on fertilization and sperm quality in pigs. Inhibitor concentrations were chosen based on the reduction of fertilization rate during preliminary dose-response experiments with cryopreserved epididymal spermatozoa. The inhibitor effects on in vitro fertilization (IVF) and sperm parameters (membrane and acrosomal integrity, motility and mitochondrial membrane potential - MMP) were evaluated using diluted fresh semen. AEBSF (100 μM), TLCK (100 μM) and TPCK (100 μM) decreased total fertilization and polyspermy rates by at least 50%. STI (5 μM) lowered total fertilization rates but not the level of polyspermy. AEBSF and TPCK reduced fertilization parameters to a similar degree using cryopreserved epididymal spermatozoa (dose-response experiment) or diluted fresh semen. Inhibition by STI was more pronounced using cryopreserved epididymal spermatozoa, whereas TLCK inhibited IVF only with diluted fresh semen. AEBSF and STI had no effect on sperm parameters, and TLCK significantly reduced motility. TPCK diminished MMP and motility and affected membrane and acrosomal integrity in a negative way. In summary, serine protease inhibitors differed in the way they reduce the fertilization rate. These results emphasize the necessity of inhibitor testing before they can be applied in fertilization studies. AEBSF and STI can be used in the future IVF studies without compromising sperm quality.