In Vitro Culture of Rhesus Macaque (Macaca mulatta) Embryos

Ramsey, Cathy; Hanna, Carol

doi:10.1007/978-1-4939-9566-0_23

Cited by 20 publications

(20 citation statements)

References 23 publications

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“…Controlled ovarian stimulation was performed on two female rhesus macaques as previously described 28,29 . Oocytes were collected and fertilized with spermatozoa by ICSI, and resulting embryos were cultured as described 30‐32 . Additional details are provided in the Supporting Methods.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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“…However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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 GnRHantagonist (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

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“…The liquid portion of the ejaculate was measured by pipetting to obtain the total volume, then transferred to a 15 mL conical tube. The coagulated plug was washed with 3 mL of TALP-HEPES (Tyrode’s Albumin, Lactate, Pyruvate with HEPES buffer, made in house) supplemented with 3 mg/mL of bovine serum albumin [ 34 ] to recover remaining spermatozoa then combined with the liquid fraction before adding additional TALP-HEPES Q.S. to 12 mL.…”

Section: Methodsmentioning

confidence: 99%

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

Houser

Ramsey

Carvalho

et al. 2021

Animals

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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.

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In Vitro Culture of Rhesus Macaque (Macaca mulatta) Embryos

Cited by 20 publications

References 23 publications

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

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

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

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

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