Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene.

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments.

Influence of substance use on male reproductive health and offspring outcomes.

The prevalence of substance use globally is rising and is highest among men of reproductive age. In Africa, and South and Central America, cannabis use disorder is most prevalent and in Eastern and South-Eastern Europe, Central America, Canada and the USA, opioid use disorder predominates. Substance use might be contributing to the ongoing global decline in male fertility, and emerging evidence has linked paternal substance use with short-term and long-term adverse effects on offspring development and outcomes. This trend is concerning given that substance use is increasing, including during the COVID-19 pandemic. Preclinical studies have shown that male preconception substance use can influence offspring brain development and neurobehaviour through epigenetic mechanisms. Additionally, human studies investigating paternal health behaviours during the prenatal period suggest that paternal tobacco, opioid, cannabis and alcohol use is associated with reduced offspring mental health, in particular hyperactivity and attention-deficit hyperactivity disorder. The potential effects of paternal substance use are areas in which to focus public health efforts and health-care provider counselling of couples or individuals interested in conceiving.

Establishing the normal range of sperm DNA fragmentation index (% DFI) for rhesus macaques.

The Sperm Chromatin Structure Assay (SCSA) is a robust test with high repeatability and precision. It is a clinically accepted assay that defines risk for infertility in men by measuring the degree of DNA fragmentation (% DFI) in sperm. The objective of this study was to adapt and validate the SCSA for rhesus macaques (Macaca mulatta) and establish a range for % DFI in fertile males. Sperm samples from two different males were used to produce a % DFI validation curve before establishing a range using additional samples from n = 11 males. Sperm labeled with acridine orange were analyzed by flow cytometry to measure green fluorescence (native or intact DNA) and red fluorescence (fragmented DNA). Data were exported to FlowJo software to determine the % DFI for each sample. DNA fragmentation ranged from 0.1 to 2.4% DFI, with a mean ± SD = 1.1 ± 0.7% DFI (validation curve optimized to R2 > 0.95). In conclusion, we were able to successfully validate the SCSA in our institution and establish the first normal range for sperm DNA fragmentation in rhesus macaques. Our study provides a quantitative baseline for future evaluations to assess macaque fertility through the SCSA test.

A Comparison of Oocyte Yield between Ultrasound-Guided and Laparoscopic Oocyte Retrieval in Rhesus Macaques.

Obtaining quality oocytes is a prerequisite for ART-based studies. Here we describe a method for transabdominal ultrasound-guided (US) oocyte retrieval in rhesus macaques (Macaca mullata) and compare it to the standard surgical approach using laparoscopy (LAP). We analyzed oocyte yield from six continuous reproductive seasons (2017-2023) that included n = 177 US-guided and n = 136 laparoscopic oocyte retrievals. While the ultrasound-guided technique retrieved significantly fewer oocytes on average (LAP: 40 ± 2 vs. US: 27 ± 1), there was no difference in the number of mature metaphase II oocytes (MII) between the two techniques (LAP: 17 ± 1 vs. US: 15 ± 1). We show that oocytes retrieved by the ultrasound-guided approach fertilize at the same rates as those obtained via the laparoscopic procedure (LAP Fert Rate: 84% ± 2% vs. US Fert Rate: 83% ± 2%). In conclusion, minimally invasive ultrasound-guided oocyte retrieval improves animal welfare while delivering equivalent numbers of mature oocytes, which are ideal for ART. Furthermore, we show that oocyte competency, as represented by fertilization rate, is not affected by retrieval technique. Therefore, the Oregon National Primate Research Center (ONPRC) has adopted the ultrasound-guided approach as the standard technique for oocyte retrieval.

Cessation of chronic delta-9-tetrahydrocannabinol use partially reverses impacts on male fertility and the sperm epigenome in rhesus macaques.

OBJECTIVE: To determine whether discontinuation of delta-9-tetrahydrocannabinol (THC) use mitigates THC-associated changes in male reproductive health using a rhesus macaque model of daily THC edible consumption. DESIGN: Research animal study. SETTING: Research institute environment. PATIENT(S): Adult male rhesus macaques (age, 8-10 years; n = 6). INTERVENTION(S): Chronic daily THC edible administration at medically and recreationally relevant contemporary doses followed by cessation of THC use. MAIN OUTCOME MEASURE(S): Testicular volume, serum male hormones, semen parameters, sperm deoxyribonucleic acid (DNA) fragmentation, seminal fluid proteomics, and whole genome bisulfite sequencing of sperm DNA. RESULT(S): Chronic THC use resulted in significant testicular atrophy, increased gonadotropin levels, decreased serum sex steroid levels, changes in seminal fluid proteome, and increased DNA fragmentation with partial recovery after discontinuation of THC use. For every increase of 1 mg/7 kg/day in THC dosing, there was a significant decrease in the total testicular volume bilaterally by 12.6 cm3 (95% confidence interval [CI], 10.6-14.5), resulting in a 59% decrease in volume. With THC abstinence, the total testicular volume increased to 73% of its original volume. Similarly, with THC exposure, there were significant decreases in the mean total testosterone and estradiol levels and a significant increase in the follicle-stimulating hormone level. With increasing THC dose, there was a significant decrease in the liquid semen ejaculate volume and weight of coagulum; however, no other significant changes in the other semen parameters were noted. After discontinuing THC use, there was a significant increase in the total serum testosterone level by 1.3 ng/mL (95% CI, 0.1-2.4) and estradiol level by 2.9 pg/mL (95% CI, 0.4-5.4), and the follicle-stimulating hormone level significantly decreased by 0.06 ng/mL (95% CI, 0.01-0.11). Seminal fluid proteome analysis revealed differential expression of proteins enriched for processes related to cellular secretion, immune response, and fibrinolysis. Whole genome bisulfite sequencing identified 23,558 CpGs differentially methylated in heavy-THC vs. pre-THC sperm, with partial restoration of methylation after discontinuation of THC use. Genes associated with altered differentially methylated regions were enriched for those involved in the development and function of the nervous system. CONCLUSION(S): This is the first study demonstrating that discontinuation of chronic THC use in rhesus macaques partially restores adverse impacts to male reproductive health, THC-associated sperm differentially methylated regions in genes important for development, and expression of proteins important for male fertility.

CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.

Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR.

Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.

Chronic exposure to delta-9-tetrahydrocannabinol impacts testicular volume and male reproductive health in rhesus macaques.

OBJECTIVE: To determine the dose-dependent effect of delta-9-tetrahydrocannabinol (THC) exposure on male testes and reproductive health in a nonhuman primate model. DESIGN: Research animal study. SETTING: Research institute. ANIMAL(S): Adult male rhesus macaques 8-10 years of age (n = 6). INTERVENTION(S): Daily edible THC at medically and recreationally relevant doses. MAIN OUTCOME MEASURE(S): Testicular volume and epididymal head width, serum levels of inhibin B, albumin, total testosterone, prolactin, follicle-stimulating hormone, estradiol, and luteinizing hormone; semen volume; and sperm motility, morphology, and concentration. RESULT(S): For each 1 mg/7 kg/day increase in THC dosing, there was a marked loss in total bilateral testicular volume of 11.8 cm3 (95% confidence interval [CI]: 8.3-15.4). In total, average bilateral testicular volume decreased by 58%. Significant dose-response decreases in mean total testosterone level by 1.49 ng/mL (95% CI: 0.83-2.15) and in estradiol level by 3.8 pg/mL (95% CI: 2.2-5.4) were observed, but significant increases in the levels of follicle-stimulating hormone by 0.06 ng/mL (95% CI: 0.02-0.10), luteinizing hormone by 0.16 ng/mL (95% CI: 0.08-0.25), and prolactin by 7.4 ng/mL (95% CI: 3.4-11.3) were observed. There were no statistically significant changes in semen parameters. CONCLUSION(S): In rhesus macaques, chronic exposure to THC resulted in significant dose-response testicular atrophy, increased serum gonadotropin levels, and decreased serum sex steroids, suggestive of primary testicular failure. Further studies are needed to determine if reversal of these observed adverse effects would occur if THC was discontinued and for validation of thefindings in a human cohort.

Proteasome inhibition triggers the formation of TRAIL receptor 2 platforms for caspase-8 activation that accumulate in the cytosol.

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario.

The effects of delta-9-tetrahydrocannabinol exposure on female menstrual cyclicity and reproductive health in rhesus macaques.

OBJECTIVE: To determine the dose-dependent effect of contemporary marijuana exposure on female menstrual cyclicity and reproductive endocrine physiology in a nonhuman primate model. DESIGN: Research animal study. SETTING: Research institute environment. ANIMALS: Adult female rhesus macaques (6-12 years of age; n = 8). INTERVENTIONS: Daily delta-9-tetrahydrocannabinol (THC) edible at medically and recreationally relevant contemporary doses. MAIN OUTCOME MEASURES: Menstrual cycle length (MCL), anti-Müllerian hormone, prolactin, basal follicle-stimulating hormone (FSH), estradiol (E2) and progesterone, luteinizing hormone (LH), and thyroid-stimulating hormone. RESULTS: The average before THC weight was 6.9 kg (standard deviation, 0.8), and at the highest THC dosing, the average weight was 7.2 kg (standard deviation, 0.8). With increasing THC dosing, MCL and FSH concentrations increased, while basal E2 concentration was stable. The average MCL concentration increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4-6.6 days). Follicle-stimulating hormone concentration increased significantly with increasing THC dose, 0.34 ng/mL for each mg/7 kg/day of THC (95% CI, 0.14-0.57 ng/mL). No significant trends were observed between THC dosing and average basal progesterone, anti-Müllerian hormone, prolactin, LH, or thyroid-stimulating hormone concentrations. CONCLUSIONS: In rhesus macaques, a dose response toward increased MCL and basal FSH concentrations but plateau of basal E2 and LH concentrations was observed with increasing THC dosing, suggesting ovulatory dysfunction. Further studies are needed to determine the effects of a longer duration of exposure and whether the significant increase in MCL and FSH concentrations results in reduced fecundity.

Effects of marijuana on reproductive health: preconception and gestational effects.

PURPOSE OF REVIEW: Recent widespread legalization changes have promoted the availability of marijuana and its increased potency and perceived safety. The limited evidence on reproductive and perinatal outcomes from marijuana exposure is enough to warrant concern and action. The objective of this review is to provide a current and relevant summary of the recent literature surrounding this topic. RECENT FINDINGS: The available published studies on the effect of marijuana exposure on reproductive health and pregnancy outcomes are conflicting. Human studies are often observational or retrospective and confounded by self-report and polysubstance use. However, the current, limited evidence suggests that marijuana use adversely affects male and female reproductive health. Additionally, prenatal marijuana exposure has been reported to be associated with an increased risk of preterm birth and small for gestational age infants. SUMMARY: With the increasing prevalence of marijuana use, there is an urgent need for evidence-driven recommendations and guidelines for couples interested in conception, affected by infertility or who are expecting. At this time, no amount of marijuana use during conception or pregnancy is known to be well tolerated and the limited available evidence suggests that the safest choice is to abstain.

Improved Training and Semen Collection Outcomes Using the Closed Box Chair for Macaques.

Collaborative semen collection in monkeys is a valuable tool in research, animal collection management, and conservation efforts. To obtain samples, monkeys are often restrained in open restraint chairs (ORC) with the "pole and collar" technique. While commonly used, this restraint is not tolerated by all individuals; some become anxious or aggressive towards the poles and people. In an effort to refine this procedure and improve welfare of the monkeys, we examined the use of a "closed box chair" (CBC), a clear, plexiglass box in which the monkey is trained to sit for sperm collection. The CBC does not require pole and collar, and although legs are secured, the arms and neck are not restrained. The use of CBCs has increased in recent years; however, there are few studies demonstrating its effects on scientific outcomes. We used positive reinforcement techniques to train 34 adult male rhesus macaques (Macaca mulatta) to provide semen samples using either the ORC or the CBC. While all CBC monkeys (n = 14) were reliably trained for this procedure, only 75% of ORC (n = 20) males completed the training (p = 0.04). It took significantly less time to train animals in the CBC than the ORC (201.0 vs. 412.4 min; p <0.001). In a controlled subset, males restrained with ORC (n = 7) produced a significantly lower ejaculatory volume than those collected by CBC (n = 10) (297.6 µL vs. 522.1 µL respectively; p = 0.04) and had a lower concentration of sperm (186.0 × 106/mL vs. 367.5 × 106/mL respectively; p = 0.017), although there were no differences with respect to sperm motility (p = 0.15). Our data suggest the closed box chair technique reduces stress on the animals while enhancing semen quality, supporting the use of the CBC as an important refinement.

Postpubertal spermatogonial stem cell transplantation restores functional sperm production in rhesus monkeys irradiated before and after puberty.

BACKGROUND: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario. OBJECTIVES: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period. MATERIALS AND METHODS: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses. RESULTS: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells. DISCUSSION AND CONCLUSION: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization.

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Metabolomics analysis of follicular fluid coupled with oocyte aspiration reveals importance of glucocorticoids in primate periovulatory follicle competency.

Gonadotropin administration during infertility treatment stimulates the growth and development of multiple ovarian follicles, yielding heterogeneous oocytes with variable capacity for fertilization, cleavage, and blastocyst formation. To determine how the intrafollicular environment affects oocyte competency, 74 individual rhesus macaque follicles were aspirated and the corresponding oocytes classified as failed to cleave, cleaved but arrested prior to blastulation, or those that formed blastocysts following in vitro fertilization. Metabolomics analysis of the follicular fluid (FF) identified 60 unique metabolites that were significantly different between embryo classifications, of which a notable increase in the intrafollicular ratio of cortisol to cortisone was observed in the blastocyst group. Immunolocalization of the glucocorticoid receptor (GR, NR3C1) revealed translocation from the cytoplasm to nucleus with oocyte maturation in vitro and, correlation to intrafollicular expression of the 11-hydroxy steroid dehydrogenases that interconvert these glucocorticoids was detected upon an ovulatory stimulus in vivo. While NR3C1 knockdown in oocytes had no effect on their maturation or fertilization, expansion of the associated cumulus granulosa cells was inhibited. Our findings indicate an important role for NR3C1 in the regulation of follicular processes via paracrine signaling. Further studies are required to define the means through which the FF cortisol:cortisone ratio determines oocyte competency.

Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis†.

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.

Restoration of functional sperm production in irradiated pubertal rhesus monkeys by spermatogonial stem cell transplantation.

BACKGROUND: In male pre-pubertal cancer patients, radiation and chemotherapy impact future fertility by eradication of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but only a small percentage of spermatozoa produced were of donor origin. Transient hormone suppression with a GnRH antagonist (GnRH-ant) enhanced spermatogenic recovery from transplanted SSCs. OBJECTIVES: To evaluate donor-derived and endogenous spermatogenic recovery after SSC transplantation into irradiated monkeys and to test whether hormone suppression around the time of transplantation facilitates spermatogenic recovery. MATERIALS AND METHODS: Testes of 15 adult rhesus monkeys were irradiated with 7 Gy and 4 months later transplanted, to one of the testes, with cryopreserved testicular cells containing SSCs from unrelated monkeys. Monkeys were either treated with GnRH-ant for 8 weeks before transplantation, GnRH-ant from 4 weeks before to 4 weeks after transplantation, or with no GnRH-ant. Tissues were harvested 10 months after transplantation. RESULTS: Two of the 15 monkeys, a control and a pre-transplantation GnRH-ant-treated, showed substantially higher levels of testicular spermatogenesis and epididymal sperm output in the transplanted side as compared to the untransplanted. Over 84% of epididymal spermatozoa on the transplanted side had the donor genotype and were capable of fertilizing eggs after intracytoplasmic sperm injection forming morulae of the donor paternal origin. Low levels of donor spermatozoa (~1%) were also identified in the epididymis of three additional monkeys. Transplantation also appeared to enhance endogenous spermatogenesis. DISCUSSION AND CONCLUSION: We confirmed that SSC transplantation can be used for restoration of fertility in male cancer survivors exposed to irradiation as a therapeutic agent. The success rate of this procedure, however, is low. The success of filling the tubules with the cell suspension, but not the GnRH-ant treatment, was related to the level of colonization by transplanted cells.

In Vitro Culture of Rhesus Macaque (Macaca mulatta) Embryos.

Production of nonhuman primate (NHP) embryos in vitro begins with recovery of gametes. Females undergo a controlled ovary stimulation to produce multiple preovulatory follicles from which oocytes may be recovered. Mature ova are subjected to in vitro fertilization (IVF) and presumptive zygotes are cultured to the intended stage of development. Essential to this practice is the culture medium unique to each step in the process. Here we describe medium preparation, oocyte recovery, in vitro fertilization, and in vitro culture (IVC) of embryos in the rhesus macaque model.

Chronically elevated androgen and/or consumption of a Western-style diet impairs oocyte quality and granulosa cell function in the nonhuman primate periovulatory follicle.

PURPOSE: To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle. METHODS: Rhesus macaques were treated using a 2-by-2 factorial design (n = 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4-5-fold increase above C) and a standard or "Western-style" diet alone (WSD) or in combination (T+WSD). Following ~ 3.5 years of treatment, females underwent controlled ovulation (COv, n = 7-10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq. RESULTS: Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33-43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (p < 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (p < 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways. CONCLUSIONS: Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.