Rhesus Macaque Sperm Research Articles

Freeze-dried primate sperm retains early reproductive potential after intracytoplasmic sperm injection

Oocytes inseminated by intracytoplasmic sperm injection using fresh ejaculated or freeze-dried rhesus macaque sperm showed similar activation, sperm aster assembly, and male-female pronuclear apposition rates.

Hyperactivated Motility in Rhesus Macaque (Macaca mulatta) Spermatozoa

Macaque spermatozoa can be capacitated according to a defined protocol and exhibit hyperactivated motility similar to that described in other species. The aim of this study was to create a method for defining hyperactivation that could be routinely used in the laboratory alongside our existing sperm motility analysis protocol. Percoll-separated macaque spermatozoa were incubated for 2 hours (37 degrees C; 5% CO(2) in air) at a concentration of 20 x 10(6)/mL in bicarbonate (36 mmol)-buffered Biggers, Whitten and Whittingham medium (BWW) containing 30 mg/mL bovine serum albumin (BSA), followed by an additional 30 minutes with (capacitated) or without (incubated) caffeine (1 mmol) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP; 1.2 mmol). One hundred and fifty progressive and hyperactivated tracks were selected from each of three monkeys. Thresholds for hyperactivation were based on the 10th (amplitude of lateral head displacement, ALH) and 90th (linearity, LIN) percentiles of the hyperactivated kinematic data set and were LIN less than or equal to 69% and ALH greater than or equal to 7.5 microM; a threshold of greater than or equal to 130 microM/s was also included for curvilinear velocity (VCL). These thresholds were 91% effective at identifying hyperactivated tracks. Capacitation of macaque spermatozoa, by the addition of caffeine and dbcAMP, resulted in a significant increase in ALH, VCL, and beat cross frequency and a significant decrease in total and progressive motility, straight line velocity, straightness, and LIN when compared to incubated spermatozoa, suggesting the expression of hyperactivated motility. Utilizing the above thresholds, hyperactivation was expressed by 5% +/- 0.8% of the incubated sperm population vs 53 +/- 3.7% of the capacitated sperm population (P < .0001). Hyperactivation was not observed when dbcAMP and caffeine were added separately and was significantly (P < .005) reduced by the addition of H-89. The results of this paper demonstrate that hyperactivation can be reliably estimated for rhesus macaque spermatozoa.

Osmotic tolerance and membrane permeability characteristics of rhesus monkey ( Macaca mulatta) spermatozoa

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 °C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7 ± 3.0 μm 3 (mean ± SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09 ± 0.30, 0.912 ± 0.27, 1.53 ± 0.53, and 1.94 ± 0.47 μm/min/atm, respectively. Cryoprotectant permeability was 1.39 ± 0.31, 2.21 ± 0.32, 3.38 ± 0.63, and 6.07 ± 1.1 (×10 −3 cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.

Presence of N-ethyl maleimide sensitive factor (NSF) on the acrosome of mammalian sperm.

We have extended previous observations to show that the ATPase N-ethyl maleimide sensitive factor (NSF) an important regulator of membrane trafficking and fusion in somatic cells, is present on bovine, murine and rhesus macaque sperm. However, NSFs main effectors, alfa- and beta-SNAP, although present in the developing acrosome, could not be detected in the mature organelle. The fact that NSF localizes mainly to the acrosome suggests that this protein, together with other factors such as rabs and SNAREs, may be a common feature in the triggering/regulation of membrane merging during the mammalian acrosome reaction.

Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa.

Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integrity to deviations from isotonicity and, in addition, that rhesus sperm motility and membrane integrity were more sensitive to hypertonic than hypotonic conditions. Mitochondrial membrane potential did not explain the lack of sperm motility observed under anisosmolal conditions in our study. Although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility.

Cryopreservation of Rhesus Macaque (Macaca mulatta) Spermatozoa and Their Functional Assessment by in Vitro Fertilization

Although spermatozoa from several species of nonhuman primates have been cryopreserved, there has been no report of success with rhesus macaque spermatozoa as judged by functional assays. Two Tris–egg yolk freezing media, TEST and TTE, which have been successfully used for cynomolgus macaque (Macaca fascicularis) spermatozoa, were compared for cryopreservation of spermatozoa from four rhesus macaques (Macaca mulatta). The postthaw motility (percentage and duration) of spermatozoa cryopreserved in TTE was much higher than that for spermatozoa cryopreserved in TEST. The function of sperm cryopreserved in TTE was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated prepubertal rhesus macaques. Of the inseminated oocytes, 82 ± 13% were fertilized and 63 ± 22 and 39 ± 21% of the resulting zygotes developed into morulae and blastocysts, respectively. These results indicate that rhesus macaque spermatozoa can be effectively cryopreserved in TTE medium. This finding will facilitate the application of in vivo and in vitro assisted reproductive technologies in this species.

Autoimmune injury of both rat testes after unilateral repeated funiculus ligation

Desiccation provides a novel spermatozoal preservation technique because it eliminates the need to store spermatozoa in liquid nitrogen, resulting in decreased opportunities for cross-contamination of samples and lower costs of spermatozoal banking, storage, and shipping. The objective of this study was to desiccate rhesus macaque spermatozoa and to evaluate the fertility following storage. Semen from four male rhesus macaques (Macaca mulatta) were collected using electroejaculation, washed through a Percoll gradient, and resuspended to 100 × 106 spermatozoa/mL in a simple vacuum drying buffer containing the disaccharide trehalose (10 mM HEPES, 5 mM KCl, 65 mMNaCl, 150 mMtrehalose, 5.7% bovine serum albumin, BSA). Cells were desiccated in 50 μl drops under vacuum (22 inHg) at ambient temperature until the water content was less than 1 g H2O/g dry weight. Initial motility was high (90–70%) and was reduced by desiccation (0%). Membrane integrity was investigated using the two fluorochromes, SYBR 14 and propidium iodide (PI, Molecular Probes, Inc.), with flow cytometry. After desiccation, 100% of the spermatozoa were stained red with PI indicating plasma membrane compromise. Samples were stored in air-tight polyvinyl plastic bags purged with N2 gas for 10 s and vacuum sealed. The samples were protected from light and either stored at room temperature (treatment 1) or at −80 °C (treatment 2). Samples were rehydrated 7–10 days post desiccation in 150 μl BWW containing 0.5% BSA and used for intracytoplasmic spermatozoa injection (ICSI) to compare fertilization and embryo development to freshly collected samples. For control embryos, one freshly ejaculated motile sperm with normal morphology was immobilized by scoring a small incision in the plasma membrane over the sperm tail before injecting into an oocyte. For the dried sperm treatments, one sperm with normal morphology was selected and scored before injecting into an oocyte. After injection, the embryos were individually cultured in CMRL medium with 10% fetal bovine serum (FBS) media on pre-plated buffalo rat liver (BRL) cells at 37 °C and 5% CO2. Fertilization was assessed at 14, 16, 22, and 24 h. Embryo development was evaluated daily from day 3 to day 11. The fertilization rate was 68%, 73%, and 45% for the control, treatment 1, and treatment 2 groups, respectively. The blastocyst rate was 40%, 5%, and 0% for control, treatments 1, and 2, respectively. Treatment group 1 had comparable fertilization rates with control group (73% vs. 68%) and was not significantly different (P < 0.05), but as development progressed, fewer embryos developed beyond the morula stage. Treatment 2 had a lower fertilization rate than control (45% vs. 68%), although not significantly different, and embryos did not develop past the morula stage. This study demonstrates that while the vacuum dried spermatozoa were immotile and had compromised plasma membrane integrity, they were capable of fertilization using ICSI and could support embryo development to the morula stage.