Formation and Dissociation of Sperm Bundles in Monotremes.

Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin; SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Sperm chromatin in beef bulls in tropical environments.

Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds.

Collection of semen by manual stimulation and ejaculate characteristics of the black flying-fox (Pteropus alecto).

Semen collection and preservation is the first step toward the development of an artificial insemination program in endangered Pteropus spp. Semen was collected by manual stimulation from a single "human-habituated" P. alecto. Manual stimulation resulted in the successful collection of motile spermatozoa on 17 of 34 attempts. The semen had a pH of 8.2 (n=2). With the exception of volume, seminal characteristics (concentration, motility, acrosome and plasma membrane status) were similar to those collected previously by electro-ejaculation. Zoo Biol 27:159-164, 2008. (c) 2008 Wiley-Liss, Inc.

Effects of cryopreservation on mitochondrial function and heterogeneity, lipid raft stability and phosphatidylserine translocation in koala (Phascolarctos cinereus) spermatozoa.

Koala sperm mitochondria were examined by cryomicroscopy using the fluorescent probe JC-1, which distinguishes high (red) and low (green) mitochondrial membrane potential (MMP). At normal body temperature, approximately 70% of live and untreated spermatozoa exhibited high MMP whereas <3% of live untreated spermatozoa exhibited low potential. A third class, in which single midpieces contained mixed mitochondrial populations, was also detected. Heterogeneity was noted in the level of MMP between individual koalas, individual spermatozoa and even between mitochondrial gyres within single midpieces. MMP of the live sperm population was not significantly affected by glycerol but was suppressed by freezing and thawing treatments. After thawing, MMP declined significantly during rewarming, especially as the temperature increased from 5 to 35 degrees C. The distribution of the ganglioside GM1 was examined using fluorescent-labelled cholera toxin B. In fresh, untreated koala spermatozoa GM1 was detected on the head and midpiece, but not on the principal piece. No significant redistribution of GM1 was observed after chilling and cryotreatment. Phosphatidylserine translocation across the plasma membrane was examined using fluorescent-labelled annexin V. Few fresh spermatozoa exhibited phosphatidylserine translocation (approximately 1%); this was not increased by chilling or cryopreservation, thus implying that cryotreatment had little effect on plasma membrane lipid asymmetry.

Simultaneous observation of DNA fragmentation and protein loss in the boar spermatozoon following application of the sperm chromatin dispersion (SCD) test.

DNA fragmentation and the nuclear protein matrix in boar spermatozoa were simultaneously assessed using a specific variant of the sperm chromatin dispersion (SCD) test that allows direct visualization of DNA and nuclear proteins under standard conditions of chemical lysis. Nuclear proteins remaining after lysis were stained with the fluorochrome 2,7-dibrom-4-hydroxy-mercury-fluorescein for specific protein staining. DNA and nuclear protein were stained in control-untreated (no lysis) and treated sperm cells (lysis), resulting in the identification of 3 cell types: type 1: nonlysed (control-untreated) cells; type 2: lysed cells showing nonfragmented DNA; and type 3: lysed cells showing fragmented DNA. DNA damage was also purposely induced by incubating the sperm in 0.015% H(2)O(2) for 48 hours at 37 degrees C; the cells were correspondingly stained for DNA fragmentation and protein. Nonlysed control sperm (type 1) nuclei showed no halos and stained strongly for protein in the postacrosomal region. Lysed spermatozoa with nonfragmented DNA (type 2) showed evidence of restricted DNA loop dispersions at the caudal extremity of the sperm head and a more homogenous but similar distribution of protein matrix in comparison with untreated spermatozoa. Lysed spermatozoa with fragmented DNA (type 3) exhibited large halos of DNA loops and a loss of the nuclear protein matrix component. Sperm cells exposed to 48 hours' incubation at 37 degrees C and then treated with the lysing agent showed a concurrent and progressive loss of nuclear protein in association with correspondingly increased levels of DNA fragmentation. Discriminant analysis of quantitative fluorescence using digital image analysis and conducted after SCD processing revealed that DNA fragmentation and protein could be evaluated in an automated system. Ninety-seven percent of the total analyzed cells were accurately classified according to previously defined cell types (1, 2, and 3). The results of the current study demonstrated a synergistic relationship between that of nuclear protein alteration and DNA damage in the boar sperm cell. The importance of abnormal nuclear protein alteration to DNA fragmentation and any related effect on fertility remains to be investigated.