Use of soluble sperm extract to improve cloning efficiency in zebrafish

Abstract

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.

Modulators of calcium signalling at fertilization

“…Activation rates of cloned horse embryos using stallion sperm extracts were significantly improved relative to other methods [ 261 ]. Recently, the use of sperm extracts for activation of SCNT reconstructed embryos in zebrafish was reported to improve the early development of parthenogenetic and cloned embryos; however, it remains uncertain if the Ca 2+ response is similar to that induced by sperm [ 262 ].…”

Sperm‐borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics

“…Injection of soluble sperm extracts into parthenogenetic embryos can improve activation and development [34–37]. Recently, researcher found that injection of sperm extract could significantly enhance the development of zebrafish embryos produced by SCNT [38]. In this study, injection of soluble protein extracts from sperm significantly increased the rate of cleavage and blastocyst formation of cloned embryos; thus, the hypothesis that sperm protein plays an important role in the development of nuclear transfer embryos was verified.…”

Reprogramming of fish somatic cells for nuclear transfer is primed by Xenopus egg extract

“…However, regeneration of fish from these highly differentiated cells requires to master nuclear transfer, a technology that is still not reliable enough in fish. Indeed, whereas nuclear transfer with embryonic donor cells yields acceptable development rates [4][5][6][7][8] , only few clones were reported to reach adulthood when the donor cell was taken from adult fish [9][10][11][12][13][14][15][16] . One hypothesis often proposed to explain the low success rate of somatic cell nuclear transfer is the chromatin reprogramming failure (reviewed in mammals [17] ).…”