Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium.
Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque.
Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection.
Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased.
Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure.
CC10; CC16; lung development; Macaca mulatta; mucin; prenatal; SCGB1A1
Errors during meiosis that affect synapsis and recombination between homologous chromosomes contribute to aneuploidy and infertility in humans. Despite the clinical relevance of these defects, we know very little about the mechanisms by which homologous chromosomes interact with one another during mammalian meiotic prophase. Further, we remain ignorant of the way in which chromosomal DNA complexes with the meiosis-specific structure that tethers homologs, the synaptonemal complex (SC), and whether specific DNA elements are necessary for this interaction.
In the present study we utilized chromatin immunoprecipitation (ChIP) and DNA sequencing to demonstrate that the axial elements of the mammalian SC are markedly enriched for a specific family of interspersed repeats, short interspersed elements (SINEs). Further, we refine the role of the repeats to specific sub-families of SINEs, B1 in mouse and AluY in old world monkey (Macaca mulatta).
Because B1 and AluY elements are the most actively retrotransposing SINEs in mice and rhesus monkeys, respectively, our observations imply that they may serve a dual function in axial element binding; i.e., as the anchoring point for the SC but possibly also as a suppressor/regulator of retrotransposition.
Meiosis; Synaptonemal complex; Chromatin Immunoprecipitation (ChIP); SINE; Synapsis; SYCP3; Mouse; Macaque
The use of therapeutic ultrasound as a contraceptive approach has involved nonhuman primates as well as rats and dogs. The current study was undertaken to determine whether this treatment could be a method for reversible contraception, using a model with testes size similar to adult humans.
Two methods of ultrasound exposure were used, either the transducer probe at the bottom of a cup filled with saline (Cup) or direct application to the surface of the scrotum (Direct). Four adult rhesus (Macaca mulatta) males with normal semen parameters were treated with therapeutic ultrasound at 2.5 W/cm(2) for 30 min. Treatment was given 3 times, one every other day on a Monday-Wednesday-Friday schedule. For each male, semen quality was evaluated a minimum of three times over several months prior to ultrasound exposure and weekly for two months following ultrasound treatment.
Semen samples from all males, regardless of exposure method, exhibited a decrease in the percentage of motile sperm following ultrasound treatment. There was an average reduction in motility of 40% the week following treatment. Similarly, curvilinear velocity and the percentage of sperm with a normally shaped flagellum were also reduced in all males following ultrasound treatment. A significant reduction in the total number of sperm in an ejaculate (total sperm count) was only observed in males that received ultrasound via the cup method. Following treatment via the cup method, males exhibited up to a 91.7% decrease in average total sperm count (n = 2). Sperm count did not approach pre-treatment levels until 8 weeks following ultrasound exposure.
The sustained reduction in sperm count, percent motility, normal morphology, and sperm vigor with the cup exposure method provides proof of principle that testicular treatment with ultrasound can be an effective contraceptive approach in humans.
Sperm morphology; Motility; Contraception; Testes; Male reproduction
The timing of the origins of fetal alcohol syndrome have been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development.
Rhesus monkey ESC lines (ORMES-6 and -7) were treated with 0, 0.01%, 0.1%, or 1.0% ethanol, 1.0% ethanol with estradiol or 0.00025% acetaldehyde with or without estradiol for 4 weeks.
Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers ( TRA-1- 81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6.
Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and -7 cell line. The morphological changes and labeling for pluripotency, proliferation and apoptosis demonstrated that ethanol affects how these early cells develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.
Primate; fetal alcohol syndrome; embryo development
To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle.
Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days.
Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin.
MAIN OUTCOME MEASURES
The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization.
RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos.
These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.
gene expression; granulosa cells; blastocyst; rhesus macaque
To determine if oral administration of a cyclooxygenase-2 (COX2) inhibitor affects oocyte nuclear maturation and fertilization in non-human primates.
Laboratory research study.
Adult female cynomolgus monkeys (Macaca fascicularis).
Monkeys received gonadotropins to stimulate multiple follicular development. An ovulatory dose of human chorionic gonadotropin (hCG) was administered either alone or concomitant with oral celecoxib, a COX2 inhibitor; oocytes were retrieved 36 hours later and exposed to sperm in vitro.
Main Outcome Measures
Oocytes were assessed for nuclear status at retrieval, resumption of meiosis in vitro, and success of in vitro fertilization.
Treatment with hCG alone yielded oocytes which were primarily at the meiosis II (MII) stage of nuclear maturation (72.9%); few oocytes were obtained at the germinal vesicle (GV) and germinal vesicle break down (GVBD) stages. Treatment with hCG and celecoxib yielded fewer mature (MII) oocytes (35.6%) and more oocytes at less mature stages when compared to oocytes from monkeys treated with hCG alone. The majority (68.3±15.9%) of MII oocytes from monkeys treated with hCG alone fertilized in vitro, compared with only 11.0±5.9% of MII oocytes from monkeys treated with hCG and celecoxib.
Oral administration of a COX2 inhibitor reduced the rate of oocyte nuclear maturation and the success of in vitro fertilization. Drugs of this class may block multiple essential steps in female reproduction and be effective contraceptives for women.
Monkey; Prostaglandin; Contraception; Ovary
Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.
stem cell; cell lineage; embryo; trophoblast
The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre–freeze (“fresh”) and post-thawed (“thawed”) samples from 5 different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, “Pap”-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.
spermatozoa; cervical mucus; hyaluronic acid; artificial insemination; monkey; cryopreservation
Glucose is important to the maturation of the oocyte and development of the embryo, while hyperglycemia results in profound reproductive and developmental consequences. However, the normal physiology of glucose in the ovary remains poorly understood. The goal of this study was to determine intra-follicular glucose dynamics during the periovulatory interval in non-human primates undergoing controlled ovarian stimulation protocols. Follicular fluid and mural granulosa cells were isolated before or up to 24 hr after an ovulatory hCG bolus, and the human granulosa-lutein cell line hGL5 was used. Intra-follicular glucose increased 3 hr after hCG, and remained at that level until 12 hr when levels decline back to pre-hCG concentrations. Pyruvate and lactate concentrations in the follicle were not strongly altered by hCG. Mural granulosa cell expression of hexokinase 1 and 2, and glucose-6-phosphate dehydrogenase mRNA decreased following hCG, while glycogen phosphorylase (liver form) increased following hCG. Glucose uptake by hGL5 cells was delayed until 24 hr following stimulation. In summary, intra-follicular glucose increases following an ovulatory stimulus and mural granulosa cells do not appear able to utilize it, sparing the glucose for the cumulus-oocyte complex.
macaque; (granulosa cells); glucose; glycolysis; luteinization
Low-density lipoprotein (LDL) extracted from hen egg yolk has recently been considered to be superior to whole egg yolk in sperm cryopreservation of various animal species. Meanwhile, there was a notion that high-density lipoprotein (HDL) in egg yolk may have a negative effect on post-thaw survival. The role of LDL and HDL in sperm cryopreservation of rhesus monkeys has not been explored. The present study evaluates their effect in comparison with egg yolk with or without the addition of permeable cryoprotectant (glycerol) on sperm cryopreservation of rhesus macaques. In addition, various additives intended to change the lipid composition of LDL–sperm membrane complex have also been tested for their effectiveness in preserving post-thaw viability. Our findings indicated that LDL is the main component in egg yolk that is responsible for its protective role for sperm cryopreservation in rhesus monkeys. Regardless of the presence or absence of glycerol, the protective role of LDL is similar to that of egg yolk and we did not observe any superiority in post-thaw survival with LDL when compared to egg yolk. Modifying the lipid composition of LDL–sperm membrane complex with the addition of cholesterol, cholesterol loaded cyclodextrin and phosphatidylcholine also did not yield any improvements in post-thaw survival; while addition of methyl-β-cyclodextrin reduced post-thaw motility. HDL plays a neutral role in sperm cryopreservation of rhesus monkeys. The present study suggests that egg yolk may still hold advantages when compared with LDL as effective components in extenders for sperm cryopreservation in rhesus monkeys.
non-human primates; sperm cryopreservation; low-density lipoprotein; high-density lipoprotein; egg yolk
Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r-hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r-hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo-matured MII oocytes and germinal vesicle (GV)- stage oocytes. Only two of 17 genes, insulin-like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r-hGH treatment group compared with other IVM treatment groups, implicating insulin-like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV-stage or MII oocytes or cumulus cells.
non-human primate; growth hormone; oocyte; gene expression
Daily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women.
Eleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg.
In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice.
BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.
biomonitoring; bisphenol A; endocrine disruption; pharmacokinetics; xenobiotic metabolism
To determine intrafollicular hormone levels and characterize mRNA expression of the IGF receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus.
Experimental animal study.
Academic medical center.
Adult rhesus macaques.
Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hr), 3, 6, 12, or 24 hr after an ovulatory hCG bolus.
Main Outcome Measure(s)
IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time RT-PCR. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and ELISA.
IGF1, IGF2 and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1 and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, 5, 6 and PAPP-A mRNAs increased following hCG, while insulin receptor and IGFBP4 mRNAs decreased following hCG treatment. IGFBP 3 and 6 and PAPP-A protein increased following hCG.
Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.
luteinization; granulosa cell; IGF; IGFBP; primate
The present study evaluated the interactions among pre-cooling, cryoprotectant, cooling, and thawing for rhesus monkey sperm using a four-way factorial design. Specifically, pre-cooling and thawing were evaluated for two conditions: slow vs. fast. Cooling was evaluated at four rates of 5, 29, 200, and 400 °C/min. The types of cryoprotectant involved combinations of egg yolk and glycerol, egg yolk and ethylene glycol, and egg yolk alone without permeable cryoprotectants or buffer alone with glycerol but without egg yolk. Our findings showed strong interactions among cryoprotectants, cooling, and thawing rates, but not pre-cooling rate, on post-thaw motility and forward progression. The optimal combination of cooling and thawing for maximum post-thaw survival depended on the types of cryoprotectant. When glycerol was used as a permeable cryoprotectant in the presence of egg yolk, slow thawing yielded similar success as fast thawing in some males. However, when glycerol was replaced with ethylene glycol for the same treatment, post-thaw motility was significantly lower in samples that were thawed slowly than those that were thawed rapidly. In the absence of permeable cryoprotectant but the presence of egg yolk, fast cooling was always favorable. On the contrary, in the absence of egg yolk but the presence of permeable cryoprotectant (glycerol), post-thaw motility was significantly reduced especially when samples were thawed slowly. Generally, fast thawing was superior to slow thawing regardless of the types of cryoprotectant or cooling rates, and glycerol in the presence of egg yolk yielded the highest post-thaw motility in all treatment groups.
cryopreservation; sperm; rhesus monkey; Macaca mulatta
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Follistatin supplementation of culture medium after fertilization improves primate embryo development, while addition during in vitro maturation decreases oocyte developmental potential.
embryo; gene expression; gene regulation; granulosa cells; oocyte development; ovary; rhesus macaque
The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.
Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization.
Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as Gαs/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment.
Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.
Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as α-fetoprotein (AFP), albumin and α1-antitrypsin (α1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the α1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, α1-AT, AFP, hepatocyte nuclear factor 3β, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and α1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 ± 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.
Although sperm cryopreservation has been studied in at least 17 non-human primate species, systematic factor optimization for any single species is lacking. Gene banking of non-human primate sperm is still in its infancy. The objective of the present study was to initiate a systematic approach to optimize the process of sperm cryopreservation for rhesus macaques, specifically, factors related to pre-freezing conditions (eg. straw freezing position, sperm concentration, sperm washing, equilibration methods, and equilibration time periods). Straw position had no effect on post-thaw motility (P = 0.193). Sperm concentration was tested in a range from 5 × 106/mL to 5 × 108/mL; post-thaw motility of sperm samples frozen at 5 × 107 cell/mL (51.0 ± 10.6%; mean ± SD) and 5 × 108 cell/mL (48.1 ± 7.3%) were higher than samples frozen at 5 × 106 cells/mL (33.0 ± 12.0%, P = 0.003). Comparison of motility immediately after thawing between samples with (51.2 ± 6.2%) and without washing (53.9 ± 6.8%) revealed no differences (P > 0.05). However, washing improved sperm forward progression within 1 h after thawing, whereas unwashed sperm retained higher post-thaw motility and progression during extended incubation (4 h) after thawing (P < 0.05). Equilibration methods (with or without pre-cooling) made no difference on post-thaw motility (P > 0.05), and the most effective equilibration time was the duration required for samples to acclimate to 4 °C prior to freezing. Evaluation and optimization of these pre-freezing conditions will help to minimize sources of injury, maximize survival, and contribute to the development of an optimized cryopreservation protocol for rhesus macaque sperm.
Cryopreservation; Sperm; Rhesus monkey; Macaca mulatta; Optimization
Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 μM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.
Energy from glycolysis but not oxidative phosphorylation is essential to support sperm motility, hyperactivated motility, and protein tyrosine phosphorylation in rhesus macaque.
acrosome reaction; sperm; sperm capacitation
The objective of this study was to use a nonhuman primate model to examine the effects of growth hormone (GH) on oocyte in vitro maturation (IVM).
Immunocytochemistry confirmed the presence of GH receptors in rhesus cumulus oocyte complexes and the cytoplasm of embryonic blastomeres. Recombinant human GH (r-hGH) was added to IVM medium and cumulus expansion, nuclear maturation, cytoplasmic maturation and embryo development were analyzed.
Cumulus expansion was highest in the presence of 1 and 10 ng/ml r-hGH. The addition of r-hGH during IVM increased the percentage of embryos progressing to at least the 9–16 cell stage. In a separate study, 100 ng/ml r-hGH was supplemented to IVM and embryo culture medium and no effect was observed.
The presence of GH receptors along with increased cumulus expansion and embryos progressing to the 9–16 cell stage supports the hypothesis that r-hGH may be involved in oocyte maturation.
Cumulus cells; Growth hormone; Non-human primate
Factors that affect sperm quality can include method of semen collection, conditions for capacitation and whether or not agglutination is present. Media and procedures for sperm washing can also impair or improve sperm function in assisted reproductive technologies. For example, the removal of seminal fluid through large volume washing is required to eliminate decapacitation activity of seminal plasma. The forces involved with centrifugation and the metabolic stress of tightly pelleting sperm during washing procedures can have deleterious results. In contrast to human sperm, sperm from the most commonly used species of nonhuman primates, rhesus and cynomolgus macaques, do not spontaneously capacitate in vitro; rather, chemical activation with dibutryl cyclic AMP and caffeine is required. Recognizing motility patterns of non-activated and activated sperm can be accomplished with simple observation. After activation, sperm agglutination sometimes occurs and can interfere with sperm binding to the zona pellucida. Because nonhuman primate oocytes require a large investment to produce and currently, each animal can be hormonally stimulated a limited number of times, it is important to have means to evaluate quality prior to using sperm from a new male for in vitro fertilization. Methods for producing live, acrosome reacted sperm may also have application for ICSI. Because many genetically valuable males are now being identified, it may be necessary to individualize sperm preparation to accommodate male-to-male variation.