Ben Colenbrander

Bio: Ben Colenbrander is an academic researcher from Utrecht University. The author has contributed to research in topics: Sperm & Oocyte. The author has an hindex of 55, co-authored 204 publications receiving 9028 citations.

Evaluation of in vitro capacitation of stallion spermatozoa.

Abstract: The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca2+-dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO2 on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca2+ ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) st...

Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane

Abstract: Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.

DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages.

Abstract: The main goal of this study was to investigate whether and at what level damage of paternal DNA influences fertilization of oocytes and early embryonic development. We hypothesized that posttesticular sperm DNA damage will only marginally affect sperm physiology due to the lack of gene expression, but that it will affect embryo development at the stage that embryo genome (including the paternal damaged DNA) expression is initiated. To test this, we artificially induced sperm DNA damage by irradiation with x- or gamma rays (doses of 0-300 Gy). Remarkably, sperm cells survived the irradiation quite well and, when compared with nonirradiated cells, sperm motility and integrity of plasma membrane, acrosome, and mitochondria were not altered by this irradiation treatment. In contrast, a highly significant logarithmic relation between irradiation dose and induced DNA damage to sperm cells was found by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and the acridin orange assay. Despite the DNA damage, irradiated sperm cells did not show any sign of apoptosis (nuclear fragmentation, depolarization of inner mitochondrial membranes, or phospholipid scrambling) and were normally capable of fertilizing oocytes, as there was no reduction in cleavage rates when compared with nonirradiated sperm samples up to irradiation doses of less than 10 Gy. Further embryonic development was completely blocked as the blastocyst rates at days 7 and 9 dropped from 28% (nonirradiated sperm) to less than 3% by greater than 2.5-Gy-irradiated sperm. This block in embryonic development was accompanied with the initiation of apoptosis after the second or third cleavage. Specific signs of apoptosis, such as nuclear fragmentation and aberrations in spindle formation, were observed in all embryos resulting from in vitro fertilization with irradiated sperm (irradiation doses >1.25 Gy). The results show that sperm DNA damage does not impair fertilization of the oocyte or completion of the first 2-3 cleavages, but blocks blastocyst formation by inducing apoptosis. Embryos produced by assisted reproductive techniques (ART) could have incorporated aberrant paternal DNA (frequently detected in sperm of sub/infertile males). Analogously, in the present work, we discuss the possibility of following embryo development of oocytes fertilized by ART through the blastocyst stage before embryo transfer into the uterus in order to reduce risks of reproductive failure.

Development of Follicles in the Mammalian Ovary

Abstract: Publisher Summary This chapter discusses the development of follicles in the mammalian ovary. The unresolved issues in follicular development are focused. Folliculogenesis culminates in the production of fully ripe, preovulatory follicles visible to the naked eye as large bulges on the surface of the ovary. Each ripe follicle contains thousands of highly differentiated cells. This complex, functional miniature organ arises from the handful of cells that constitute a simple primordial follicle, a structure so small that it is invisible at the lower magnifications of a light microscope. All regulatory influences can only permit or prevent cells from completing the full maturation process; they cannot change the course of differentiation. A plethora of modulating influences act as permissive inducers, impeding or propelling the committed follicular cells through the process of clonal expansion. As each follicle progresses through its program of limited clonal expansion and maturation, its cells proliferate more and more rapidly. With every passing generation, the proliferative potential of the granulosa and theca cells continues to diminish, while the state of maturation increases.