Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat

Tanaka, Takeo; Nakao, Satohiro; Mikoda, Nobuyuki; Yamaga, Katsuma; Maeda, Ryusei; Tsuchiyama, Shuuji; Nakatsukasa, Ena; Nakagata, Naomi

doi:10.1038/s41684-022-01053-5

Cited by 9 publications

(12 citation statements)

References 60 publications

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“…Rats have many advantages as a model species because of the vast literature on rat sex differences, their greater utility in some models of human disease, and their larger size [19][20][21][22]. Transgenesis is used increasingly in rats, and rat genomic and epigenomic resources are being rapidly developed [23][24][25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

A “Four Core Genotypes” rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

Arnold¹,

Chen²,

Grzybowski³

et al. 2023

Preprint

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Background: We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods: XY rats were produced with an autosomal transgene of Sry, the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results: We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion: The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

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Section: Introductionmentioning

confidence: 99%

A “Four Core Genotypes” rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

Arnold¹,

Chen²,

Grzybowski³

et al. 2023

Preprint

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“…Transgenic animal production is predominantly predicated by the quality of the oocytes, which supply the coordinating factors fundamental to early embryonic development (Schultz & Heyner, 1992). Therefore, alterations for transgenic animal production are performed in vitro (Takeo et al, 2022). However, oocytes are considerably vulnerable to harmful external physicochemical factors (high oxygen, temperature, and toxins) and accumulate reactive oxygen species (ROS) during in vitro maturation (IVM) and culture (IVC), because they lack the natural antioxidant protection that is present in vivo.…”

Section: Introductionmentioning

confidence: 99%

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1‐induced enhancement of mitochondrial activity and alleviation of proapoptotic events

He,

Liu,

Wang

et al. 2023

Reprod Domestic Animals

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Notoginsenoside R1 (NGR1), derived from the Panax notoginseng root and rhizome, exhibits diverse pharmacological influences on the brain, neurons, and osteoblasts, such as antioxidant effects, mitochondrial function protection, energy metabolism regulation, and inhibition of oxygen radicals, apoptosis, and cellular autophagy. However, its effect on early porcine embryonic development remains unclear. Therefore, we investigated NGR1's effects on blastocyst quality, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial function, and embryonic development‐related gene expression in porcine embryos by introducing NGR1 during the in vitro culture (IVC) of early porcine embryos. Our results indicate that an addition of 1 μM NGR1 significantly increased glutathione (GSH) levels, blastocyst formation rate, and total cell number and proliferation capacity; decreased ROS levels and apoptosis rates in orphan‐activated porcine embryos; and improved intracellular mitochondrial distribution, enhanced membrane potential, and reduced autophagy. In addition, pluripotency‐related factor levels were elevated (NANOG and octamer‐binding transcription factor 4 [OCT4]), antioxidant‐related genes were upregulated (nuclear factor‐erythroid 2‐related factor 2 [NRF2]), and apoptosis‐ (caspase 3 [CAS3]) and autophagy‐related genes (light chain 3 [LC3B]) were downregulated. These results indicate that NGR1 can enhance early porcine embryonic development by protecting mitochondrial function.

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“…Recently, we designed a successful protocol for rat sperm cryopreservation and subsequent IVF [ 17 , 18 ]. Cryopreserved rat sperm can be permanently preserved in a liquid nitrogen tank, and animals can be quickly reproduced using IVF and embryo transfer techniques.…”

mentioning

confidence: 99%

“…Several recent reports have shown that high fertilization rates can be achieved even if the sperm is inseminated after preincubation for 15–60 min (before the completion of capacitation) [ 7 , 16 ]. In addition, we developed a successful protocol for IVF using cryopreserved rat sperm and used 2-h preincubation time for frozen–thawed sperm before insemination is generally adopted [ 17 , 18 ].…”

mentioning

confidence: 99%

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Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes

NAKAGATA,

NAKAO,

MIKODA

et al. 2024

Journal of Reproduction and Development

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Genetically modified rats are valuable models in human disease research. We recently developed an improved system for rat sperm cryopreservation and in vitro fertilization (IVF) that facilitates the efficient production and preservation of genetically modified rats. In the IVF procedure performed using frozen–thawed rat sperm, the IVF schedule is fixed to ensure timely hormone administration and oocyte collection. To enhance the flexibility of the IVF schedule, possible periods of postovulated rat oocytes with normal fertility and developmental abilities should be determined. Therefore, in this study, we examined the fertilization and developmental ability of incubated oocytes 1–13 h after oocyte collection at 9:00 AM. The fertilization rate decreased 7 h after oocyte collection, and abnormally fertilized oocytes appeared 10 h after oocyte collection. The developmental rate also decreased 7 h after oocyte collection; however, live pups were obtained from oocytes 12 h after oocyte collection. In summary, ovulated rat oocytes exhibited a high developmental ability after IVF for up to 4 h after oocyte collection.

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Optimized protocols for sperm cryopreservation and in vitro fertilization in the rat

Cited by 9 publications

References 60 publications

A “Four Core Genotypes” rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

A “Four Core Genotypes” rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

Improving the developmental competences of porcine parthenogenetic embryos by Notoginsenoside R1‐induced enhancement of mitochondrial activity and alleviation of proapoptotic events

Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes

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