X-ray microscopy of human spermatozoa shows change of mitochondrial morphology after capacitation

Using X-ray microscopy two morphologically distinct states were observed of the human spermatozoan mitochondria: (i) compact and tightly wrapped around the axoneme, and (ii) morphologically transformed, i.e. with circular areas of high X-ray transmission, either loosely wrapped around the axoneme or distended. The spermatozoa were examined at two stages of their post-ejaculation maturation process, i.e. as present in fresh ejaculated semen and after in-vitro capacitation. X-ray microscopy allowed sample preparation that was as simple as for conventional light microscopy whilst giving high resolution (30 nm) imaging of samples in liquid media compatible with the requirements of live biological specimens. The specimens were not fixed, stained or metal coated. These features make X-ray microscopy useful in the study of cells, particularly cells in suspension. The relative frequencies of the two morphological states of the mitochondria in seminal plasma and after in-vitro capacitation were compared. In seminal plasma, almost all spermatozoa had compact and tightly wrapped mitochondria. After harvesting by swim-up technique, an increase in the morphologically transformed state had occurred. However, the greatest increase in the morphologically transformed state occurred when the sample had been incubated under capacitating conditions. In this case almost all spermatozoa had morphologically transformed mitochondria.