In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network.

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time-period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post-thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital® (SV) technology, the first commercialized production line of SpermVital® (C) and by conventional procedure applying Biladyl® extender (B). Post-thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital® technology is improved and is more efficient in conserving post-thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.

Reproductive performance of immobilized cryopreserved bovine semen used for timed artificial insemination.

The SpermVital® technology comprises embedding of spermatozoa within an alginate gel to facilitate release of sperm cells over a prolonged period in utero after AI. The aim of this study was to examine whether the survival time of spermatozoa is extended when applying this immobilization technology in combination with cryopreservation. Sperm cell survival (acrosome and plasma membrane integrity) was studied in vitro for 48 hr at physiological temperature. One dose of SpermVital® (SV) semen was compared with single doses of Biladyl® (B) processed semen as well as double doses of B (B double). B double was obtained by adding a second B dose the following day, thereby mimicking double AI. Furthermore, reproductive performance applying single early timed AI (TAI) with SV following oestrus synchronization was studied in a field trial. Double insemination (TAI on two consecutive days) with B semen served as control. Number of acrosome-intact live sperm cells decreased over time in vitro for all treatments (p < .05). There was no difference between SV sperm cell survival and B double after 24 hr (p > .05). However, after 48 hr, SV sperm cell survival was higher than B double (p < .05). Moreover, multivariate analysis showed that the outcome of single early TAI with SV was not significantly different from B double (p > .05). Likelihood of pregnancy and calving in the heifer group was higher than in the cow group (p < .05). These results imply that spermatozoa immobilized in alginate gel have prolonged survival.

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability.

c-Myb is an essential hematopoietic transcription factor that controls proliferation and differentiation of progenitors during blood cell development. Whereas sumoylation of the C-terminal regulatory domain (CRD) is known to have a major impact on the activity of c-Myb, no role for noncovalent binding of small ubiquitin-like modifier (SUMO) to c-Myb has been described. Based on the consensus SUMO-interacting motif (SIM), we identified and examined putative SIMs in human c-Myb. Interaction and reporter assays showed that the SIM in the in the transactivation domain of c-Myb (V(267)NIV) is functional. This motif is necessary for c-Myb to be able to interact noncovalently with SUMO, preferentially SUMO2/3. Destroying the SUMO-binding properties by mutation resulted in a large increase in the transactivation potential of c-Myb. Mutational analysis and overexpression of conjugation-defective SUMO argued against intramolecular repression caused by sumoylated CRD and in favor of SUMO-dependent repression in trans. Using both a myeloid cell line-based assay and a primary hematopoietic cell assay, we addressed the transforming abilities of SUMO binding and conjugation mutants. Interestingly, only loss of SUMO binding, and not SUMO conjugation, enhanced the myeloid transformational potential of c-Myb. c-Myb with the SIM mutated conferred a higher proliferative ability than the wild-type and caused an effective differentiation block. This establishes SUMO binding as a mechanism involved in modulating the transactivation activity of c-Myb, and responsible for keeping the transforming potential of the oncoprotein in check.

FLASH acts as a co-activator of the transcription factor c-Myb and localizes to active RNA polymerase II foci.

The c-Myb oncoprotein is a DNA-binding transcription factor with a key role in early stages of hematopoiesis. To expand our knowledge of partners cooperating with c-Myb, we performed a yeast two-hybrid screening with full-length c-Myb as bait. Here, we report FLICE-associated huge protein (FLASH)/CASP8AP2 as a novel Myb-interacting protein. We show that FLASH interacts with the DNA-binding domain of c-Myb and enhances c-Myb-dependent reporter activity and expression of endogenous c-Myb target genes. Chromatin immunoprecipitation assays revealed that FLASH and c-Myb both associate with the MYC promoter region as well as with the intronic enhancer of the c-Myb target gene ADA. Furthermore, siRNA knock-down of FLASH or c-Myb both result in a reduction of MYC and ADA expression. The co-activator effect is mediated through the C-terminal part of FLASH, which binds c-Myb. The FLASH-induced enhancement is comparable with the increase seen with the c-Myb co-activator p300. We find FLASH localized in discrete nuclear speckles in several cell lines, co-localized with c-Myb in active RNA polymerase II foci. These results imply a novel molecular mechanism of regulation of c-Myb activity. We propose that c-Myb cooperates with FLASH in foci associated with active RNA polymerase II, leading to enhancement of Myb-dependent gene activation.