This study examined molecular mechanisms involved in the activation of motility in spermatozoa from the cauda epididymidis of rats. A 1.05-fold dilution of semen from the cauda epididymidis with 300 mmol sucrose l −1 did not activate motility in spermatozoa. Addition of dibutyryl cAMP, pentoxifylline or Ca 2+ to the sucrose activated motility in the short term (< 30-60 min). A fivefold dilution of semen from the cauda epididymidis with a modified Tyrode's medium (BWW) activated and sustained vigorous motility that could not be attenuated with kinase inhibitors. This motility was associated with a transient increase in intracellular cAMP during the first 60 s of activation. Lower motility was activated in Ca 2+ -deficient media but this was not associated with an increase in cAMP. A fivefold dilution with plasma from the cauda epididymidis did not activate motility. The addition of Ca 2+ to the sucrose induced an increase in cAMP of similar duration but lower magnitude to that associated with dilution in BWW. The results from this study indicate that the cAMP and Ca 2+ signal transduction pathways are involved in activation of sperm motility, and that the increase in intracellular cAMP in rat spermatozoa from the cauda epididymidis undergoing motility activation is Ca 2+ -dependent. This is the first study to report a Ca 2+ -dependent increase in cAMP associated with motility activation in immotile mammalian spermatozoa. In light of these data, a model is proposed whereby cAMP and Ca 2+ act as synarchic messengers, initiating a signal transduction cascade, which is independent of protein kinase A-mediated phosphorylation of flagella proteins in immotile spermatozoa from the cauda epididymidis.

Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.

The metabolism, rate of intracellular accumulation of sugars, motility and ultrastructure of ejaculated tammar sperm were impaired by swim-up into artificial media, particularly when the cells were subsequently exposed to N-acetyl-D-glucosamine (NAG). The inclusion of hyaluronate, serum albumin, catalase or Desferal in swim-up media helped prevent deterioration of sperm motility, but failed to prevent detrimental NAG-induced metabolic and ultrastructural changes. However, the sperm were unavoidably diluted during swim-up into artificial media and their behavioural properties were modified by dilution. Thus, sperm collected from the cauda epididymidis were immotile and their rate of oxygen uptake was low in undiluted caudal epididymal semen (CES). Nevertheless, these sperm were viable, and vigorous motility was induced by 5- to 50-fold dilution in Krebs-Ringer phosphate (KRP). Sperm respiration also dramatically increased with moderate dilution (5- or 15-fold) in KRP, but decreased again at higher rates (50-fold). This suggested that motility and the metabolic properties of tammar sperm are modified both by dilution and on leaving the suppressing conditions of the epididymis. Diluted tammar epididymal sperm also displayed a Pasteur effect, but rapidly lost capacity for motility in an oxygen-depleted atmosphere. It was concluded hat swim-up procedures compromise ejaculated tammar sperm by promoting dilution-induced changes. This may alter the permeability of the membrane with loss of the enzymes that process the ammonia generated during the metabolism of NAG in seminal plasma. Subsequent exposure to NAG further promotes ultrastructural damage culminating in loss of viability.

Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acrosome onto the nucleus during sperm formation, and for the stability of the nuclear membrane during fertilization. For the first time, subacrosomal proteins have been isolated from a marsupial species, the tammar wallaby (Macropus eugenii), using a method developed in our laboratory. Whole ejaculated spermatozoa were fractionated into head and tail sections by ultrasonication to extract subacrosomal proteins. The heads (> 95% purity) were then isolated from tail sections using centrifugation with a three-step discontinuous sucrose gradient (35, 68 and 75% (w/v). The heads were treated with 0.1% (v/v) Triton X-100 which stripped off the acrosome, but not the subacrosomal proteins, from the head. The proteins were finally extracted by 100 mmol NaOH l-1. Four prominent subacrosomal polypeptides, with molecular masses of 45, 38, 33 and 29 kDa, were recognized from the SDS-PAGE gel. The localization of these polypeptides (particularly the 45 kDa polypeptide) was confirmed by fluorescent and immunogold labelling with polyclonal antibodies raised in mice against the obtained polypeptides. In wallaby testes, the 45 kDa polypeptide was detected as early as at the step 3 spermatid and was mainly associated with the membrane of the newly formed acrosome vesicle. This polypeptide was also found on the acrosomal membrane of all older spermatids. The 45 kDa polypeptide was found on the acrosomal region of the spermatozoa collected from the caput, corpus and cauda of the epididymis. The similarity of the sperm anatomy of the tammar wallaby with that of other marsupials, such as the brushtail possum, implies that this procedure could be applied effectively to other marsupial species with minor modification.