Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA

Abstract

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

Production of mouse offspring from zygotes fertilized with freeze-dried spermatids

“…It is known that the success rate of offspring production decreases signi cantly when FD spermatozoa are used compared to the use of intact spermatozoa (average birth rate from fresh spermatozoa were around 60%, whereas from FD spermatozoa were around 20%) 6,34 . It is also known that the success rate of offspring production decreases largely when fresh spermatids are used compared to when fresh spermatozoa are used (average birth rate from fresh spermatozoa were around 60%, whereas from fresh round spermatid were around 20%) 12,13 . Thus, the synergistic negative effect of both methods (FD treatment and the use of spermatids) would have greatly reduced the success rate of FD spermatids.…”

“…To collect spermatids, the seminiferous tubules of the testes were minced using sharp scissors, as described previously 13 ; the only difference was that the cells were suspended in HEPES-buffered CZB medium 12 . In order to collect spermatozoa, we rst collected epididymides and cut their ducts using sharp scissors, as described previously 28 .…”

“…In such cases, it may be possible to collect spermatids from the testes, in which haploid male germ cells are found, before the completion of spermiogenesis. Importantly, even though spermatids cannot fertilize oocytes by themselves, when spermatids are arti cially injected into oocytes, healthy offspring can be obtained 12,13 . Although the use of this method results in lower birth rates than when mature spermatozoa are used, it suggests that spermatids are important components for the preservation of the genetic material of infertile or immature male animals 14 .…”

Oocyte activation during round spermatid injection: state of the art

“…It was demonstrated that human spermatozoa devoid of PLCζ1 failed to induce Ca 2+ release and were unable to initiate the first step of embryo development (Yoon et al, 2008), while injection of recombinant human PLCζ1 induced intracellular calcium oscillations and oocyte activation in mouse and human oocytes (Swann et al, 2012;Yoon et al, 2012;Yamaguchi et al, 2017). Therefore, PLCζ has been used to induce Ca 2+ oscillations for assisted oocyte activation or artificial oocyte activation (AOA) (Amdani et al, 2013;Anifandis et al, 2019;Hirose et al, 2020). Barberan et al (2022) reviewed the advantages and disadvantages of using PLCζ1 as a diagnostic marker or therapeutic molecule in human ICSI procedures.…”

Strategies to Identify Genetic Variants Causing Infertility

“…In particular, genetic defects in oocytes or early embryos can be compensated by introducing exogenous protein or synthetic mRNA (sometimes called complementary RNA, cRNA). For example, defects in egg activation due to mutations in the sperm-borne protein phospholipase C ζ1 (PLCZ1) can be rescued by recombinant protein or cRNA [91][92][93]. Proof-of-principle for the oocyte cRNA correction approach has also been reported for mutations in WEE2,…”