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.     

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.     

Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta)

Kisspeptin‐Kiss1R signalling in mammals has been implicated as an integral part of the reproductive cascade. Kisspeptinergic neurons upstream of GnRH neurons are involved in the activation of the hypothalamic GnRH pulse generator during pubertal onset. Thus, the major research focus has been on the central effects of kisspeptin. The demonstration of the presence of KissR expression in human testes suggests additional unknown actions of kisspeptin‐KISS1R signalling at the distal component of the male reproductive axis. Here we explored the impact of kisspeptin at the testis in the adult male rhesus monkey. We employed the clamped monkey model to assess the intratesticular actions of kisspeptin. Plasma testosterone and LH levels were monitored in four adult male monkeys. The peripheral administration of human kisspeptin‐10 (50 μg, iv bolus) caused a single LH pulse, which was followed by a robust increase in plasma testosterone levels sustained for at least 180 min. This response was abolished when kisspeptin was administered to GnRH receptor antagonist (acyline) pre‐treated animals. However, kisspeptin administration significantly (P < 0.005) elevated hCG‐stimulated testosterone levels in acyline pre‐treated monkeys when compared with saline+ hCG treatment. These results revealed a novel peripheral facet of kisspeptin signalling.     

Cryopreservation of cynomolgus monkey （Macacafascicularis） spermatozoa in a chemically defined extender

AIM: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. METHODS: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. RESULTS: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. CONCLUSION: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.     

cDNA cloning, protein structure modeling of rhesus monkey (Macaca mulatta) prothrombin

OBJECTIVE: Hemorrhagic diseases have been considered to be one of the main causes of xenotransplantation failure. To explore the role of the rhesus monkey (Macaca mulatta) coagulation system in a pig-to-human xenotransplantation model, we primarily studied the full-length cDNA and the three-dimensional (3-D) structures of the important coagulation factor-prothrombin of the rhesus. MATERIALS AND METHODS: The full-length cDNA of rhesus prothrombin was obtained by rhesus monkey liver cDNA library screening and a 5'RACE technique. The 3-D protein structure was modeled using the SWISS-MODEL program. The important macromolecular interaction sites were compared with human and porcine prothrombin. RESULTS: At first, the full-length rhesus prothrombin cDNA was cloned; the sequence was submitted to the Genebank (accession number: EF057490). The full-length cDNA is 2029 bp, with a complete open reading frame of 1884 bp, coding 626 amino acids. The deduced protein sequence contains a signal peptide, propeptide, Gla domain, two Kringle domains, and a Trypsin domain. The nucleotide similarities of rhesus-human and rhesus-porcine genes are 95.64% and 86.14%, and those of the amino acids, 94.51% and 82.82%, respectively. Some important functional sites, such as the catalytic triad DHS, RGD, Na+ -binding, and the carboxylase-binding site, are highly conserved. However, among the three species some variations are observed in potential N-glycosylation, O-glycosylation, phosphorylation, cleavage site, FXa-binding sites. Especially, the autolysis loop shows great differences in both amino acid sequence and 3-D model. CONCLUSIONS: The great similarity of prothrombin among rhesus, human, and porcine confirmed the great value of the pig-to-rhesus xenotransplantation model. The variation especially in some important recognition sites between rhesus and pig would vary the binding affinity of enzymes to xenosubstrates and their reaction velocities in postoperative coagulation processes, which would be one possible reason for the disordered regulation of clotting seen during the xenorejection in animal models.     

Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO(2) in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO(3). Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 micromol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochemically examined by means of anti-phosphotyrosine (alpha-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P < .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P < .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P < .0001) and a significant increase in the intensity of merocyanine fluorescence (P < .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.     

Seasonal changes in steroidogenesis in the testis of the rhesus monkey (Macaca mulatta)

To investigate the influence of seasonal changes from breeding to nonbreeding season on testicular steroidogenesis in the adult rhesus monkey, two animals were hemiorchidectomized in November and the second testis of each was removed the following April. Intratesticular steroid concentrations related to the formation of testosterone from pregnenolone were measured by radioimmunoassay. Related steroidogenic enzyme activities were also assayed and dynamic studies of the bioconversion of radiolabeled precursor steroids by testicular microsomes were performed. Testosterone was synthesized from pregnenolone through both dehydroepiandrosterone and 17 alpha-hydroxyprogesterone. No distinct differences were found in the testosterone biosynthetic pathway between testes in breeding and nonbreeding seasons. In the breeding season, intratesticular testosterone concentrations, the sum of intratesticular steroids measured, and the ratio of testosterone to the steroid sum were increased when compared to those in the nonbreeding season. No major seasonal changes in microsome enzyme activities were found, although enzyme activities, except for delta 5-3 beta-hydroxysteroid dehydrogenase for C21-steroids, were slightly higher in the breeding season. We conclude that increased testosterone production in the testis during the breeding season is due primarily to increased pregnenolone supply from Leydig cell mitochondria and, to a lesser extent, to an enhanced pregnenolone bioconversion to testosterone in the microsomes.     

Characterization and androgen dependence of specific proteins in the epididymis of adult rhesus monkey (Macaca mulatta)

The androgen dependence and regional distribution of specific epididymal proteins have been investigated in the adult rhesus monkey (Macaca mulatta). The protein pattern of the epididymal cytosol was studied in intact, castrated, and testosterone-treated castrated monkeys by polyacrylamide gel electrophoresis (PAGE) and electrofocusing. At least five distinct protein components of the epididymal cytosol were androgen-sensitive. The PAGE of cytosol from various portions of the epididymis showed that whereas four protein components were uniformly distributed in the caput, corpus, and caudal segments, one of the androgen-dependent proteins was found to be associated with the caudal portion of the epididymis. Electrofocusing of epididymal cytosol also confirmed the presence of five androgen-dependent proteins in the adult monkey. The isoelectric points of these proteins were shown to range between 5.6 and 6.5, and the molecular weights were found to range between 15,000 and 61,000.     

Progesterone-induced calcium influx in cynomolgus monkey (Macaca fascicularis) spermatozoa

For in vitro capacitation to occur in cynomolgus monkey (Macaca fascicularis) spermatozoa, there is an absolute requirement for exogenous stimulation with the sperm activators, caffeine (1 mM) and db-cyclic adenosine monophosphate (dbcAMP) (1 mM), which are known to induce capacitation-related hyperactivated motility. Tyrosine phosphorylation of sperm tail proteins is an integral component of this caffeine- and dbcAMP-stimulated hyperactivated motility. In both capacitated and noncapacitated human spermatozoa, progesterone (P4) has been reported to elicit an immediate, potent increase in intracellular calcium ion concentrations [Ca2+]i. The objective of this study was to examine the effects of progesterone on requisite events in macaque fertilization, including [Ca2+]i, hyperactivated motility, and the concomitant tyrosine phosphorylation of sperm tail (STTP) proteins after treatment with caffeine and dbcAMP. The effect of 1 microM of progesterone on [Ca2+]i was determined by spectrofluorometry with the fluorescent indicator, fura-2/AM, on hyperactivated motility using computer analysis (HTM-IVOS) with the sorting criteria lateral head amplitude (> or = 8.0 microm), curvilinear velocity (> or = 150 microm/s), linearity (< or = 60%), and on STTP by immunocytochemistry. The results showed that progesterone elicited a significant increase in [Ca2+]i in caffeine- and dbcAMP-activated macaque sperm with maximal stimulation at 30 minutes after activation. The response in nonactivated sperm was dramatically reduced compared with the response in activated sperm. Basal [Ca2+]i increased as a function of time in both activated and nonactivated control sperm although basal levels were significantly increased in activated sperm. Progesterone stimulation resulted in a small but significant increase in both hyperactivation and STTP when sperm were first pretreated with caffeine and dbcAMP. Our results provide evidence that macaque sperm activation with caffeine and dbcAMP is required for a progesterone-elicited response, which results in calcium influx, hyperactivated motility, and sperm tail tyrosine phosphorylation.     

Seasonality in endocrine and exocrine testicular function of the adult rhesus monkey (Macaca mulatta) maintained in a controlled laboratory environment

Testicular exocrine and endocrine function was monitored in adult male rhesus monkeys maintained for up to 4 years in a controlled environment isolated from seasonal changes in light, humidity and temperature and from female animals. Marked circannual variations were found in both functions. Spermatogenesis and exocrine function were maximal in the autumn and winter months as indicated by histological studies and the measurement of testicular volume, frequency of spontaneous and provoked ejaculations and in sperm output. The serum level and metabolic clearance rate and production rate of testosterone were also maximal during this period as were the serum concentrations of dihydrotestosterone, cortisol and biologically active luteinizing hormone. By contrast the serum prolactin and dehydroepiandrosterone levels showed an inverse pattern, achieving their highest levels in spring, during the period of reduced testicular function. Since such marked circannual variations in testicular and pituitary functions persist in monkeys isolated from external environmental influences, the existence of an inherent regulatory mechanism can be postulated. Such distinct variations in testicular function must be taken into consideration when using the male rhesus monkey as an experimental model for human reproductive function.     

Application of three-dimensional culture systems to study mammalian spermatogenesis,with an emphasis on the rhesus monkey (Macaca mulatta)

In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.     

Zinc in the seminal vesicle and cranial and caudal prostate of rhesus monkey (Macaca mulatta): testicular regulation and subcellular distribution

Zinc concentration and its subcellular distribution in the seminal vesicle and cranial and caudal prostate of adult rhesus monkey was determined by atomic absorption spectroscopy. Its concentration (microgram/mg protein) was maximum in the caudal lobe of prostate (5.7 micrograms) followed by cranial prostate (2.8 micrograms) and seminal vesicle (1.26 microgram). Analysis of subcellular fractions revealed that zinc concentration was highest in the microsomal fraction constituting about 50% of total zinc in the two lobes of prostate (cranial: 5 micrograms; caudal: 17 micrograms/mg protein). A significant reduction occurred in their zinc content following castration. The possible role of zinc in the accessory sex glands is discussed.     

Reference values of clinical chemistry and hematology parameters in rhesus monkeys (Macaca mulatta)

Chen Y, Qin S, Ding Y, Wei L, Zhang J, Li H, Bu H, Lu Y, Cheng J. Reference values of clinical chemistry and hematology parameters in rhesus monkeys (Macaca mulatta).
Xenotransplantation 2009; 16: 496–501. © 2009 John Wiley & Sons A/S. Abstract: Background: Rhesus monkey models are valuable to the studies of human biology. Reference values for clinical chemistry and hematology parameters of rhesus monkeys are required for proper data interpretation. Methods: Whole blood was collected from 36 healthy Chinese rhesus monkeys (Macaca mulatta) of either sex, 3 to 5 yr old. Routine chemistry and hematology parameters, and some special coagulation parameters including thromboelastograph and activities of coagulation factors were tested. Results and conclusion: We presented here the baseline values of clinical chemistry and hematology parameters in normal Chinese rhesus monkeys. These data may provide valuable information for veterinarians and investigators using rhesus monkeys in experimental studies.     

The vertebral cartilaginous endplate of the preadult rhesus monkey (Macaca mulatta). A correlative scanning electron-and light-microscopic study

The microanatomy of the preadult rhesus monkey (Macaca mulatta) vertebral cartilaginous endplate was investigated with scanning electron microscopy and light microscopy. The plate contains three zones: an outer noncalcified zone adjacent to the intervertebral disc, a middle zone of acellular islands of calcification surrounded by cellular hyaline cartilage, and an inner zone resembling a cartilaginous growth plate. Fibers from the annulus fibrosus pass through an outer zone into the middle zone. Trabecular bone of a developing annular epiphysis is present in the periphery of the plate. Vertical columns of cartilage traverse the nuclear region of the endplate and occasionally surround blood vessels. These columns and their associated blood vessels probably represent notochordal and fetal blood vessel remnants.     

Effect of kisspeptin challenge on testosterone and inhibin secretion from in vitro testicular tissue of adult male rhesus monkey (Macaca mulatta)

Kisspeptin expression has been found in gonads but a direct role of kisspeptin in reproduction is not known. The objective of this study was to find a dose and time related effect of kisspeptin on testicular hormones secretion of adult male rhesus monkey (n = 5). Kisspeptin (1, 10, 100, 1000 pm ) was incubated to a culture of testes (100 mg fragments) of male rhesus monkey and medium for hormone (testosterone and inhibin) measurement was collected after 30, 60 and 120 min. 10 IU hCG (180 min) and 50 ng FSH (60 and 120 min) were incubated to the culture for checking testicular cells ability to secrete hormones in vitro. Kisspeptin did not significantly (P < 0.05) increase the testosterone and inhibin levels at any dose. However, one way anova at pooled doses showed an increase in testosterone levels and paired t‐test at pooled doses showed inhibin decrease after 120 min of incubation suggesting an independent effect of time. hCG and FSH significantly (P < 0.05) increased hormone concentration compared to the basal groups. We concluded that kisspeptin has no role in testicular regulation related to testosterone and inhibin release but kisspeptin may have other roles in testicular regulation.     

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta)

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.     

Ultrastructure of monkey (Macaca radiata) spermatozoa: effect of gossypol in vivo

Summary The present study examines the ultrastructure of ejaculated spermatoza from bonnet monkey, Macaca radiata under noraml conditions, with gossypol treatment and during recovery from such treatment. Monkeys were fed orally with gossypol acetic acid (GAA) for 3 months (4 mg/monkey/5 days a weak). Semen samples collected by electroejaculation, and the spermatozoa were examined using both light and electron microscopy. The degree of motility was also noted by Kalla et al. [12]. Ejaculated spermatoza were immotile 90 days after GAA treatment, but little evidence for any abnormality in the spermatozoa could be seen by light microscopy. Some ultrastructural changes were observed, but not to the extent previously reported in spermatozoa of Macaca fascicularis [23]. After termination of treatment, semen samples were obtained every 5th day until sperm count and motility recovered to the normal level. After 90 days only a small proportion of spermatozoa showed abnormal structure. We conclude that in a subhuman animal model gossypol induced effects on sperm motility and morphology are reversible.