Oocyte activation after intracytoplasmic injection with sperm frozen without cryoprotectants results in live offspring from inbred and hybrid mouse strains

Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr 2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P < 0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P < 0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits

Following in vitro fertilization and culture, both fresh (A1) and vitrified oocytes in 0.1 M trehalose (B1) were capable of developing to the two-cell (A2; B2), four-cell (A3; B3) and blastocyst (A4; B4) stages, respectively.

<p>Furthermore, blastocysts were able to hatch (A5; B5) without chemical or mechanical assistance and some were used to derive embryonic stem cells.</p

Survival, and live offspring rates for vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05). Oocyte and live offspring data collected from 5 and 2 replicates respectively.</p

Survival, two-cell and blastocyst rates for vitrified mouse oocytes following IVF.

*<p>Fisher's test significantly lower than other treatments (P&lt;0.05);</p>**<p>Fisher's test significantly higher than other treatments within the same column (P&lt;0.001). IVF Data collected from 3 replicates.</p

Karyotype analysis of ES cells and live offspring derived from vitrified oocytes.

<p>A) Karyotype analysis performed on ViO-ES9 cells revealed a number of abnormalities. The male cell line has a chromosome count of 46. B) Karyotype analysis performed on the mice that were generated from vitrified oocytes revealed normal karyotypes, a representative karyotype for one of the male mice shows a normal 40 XY chromosome count with no abnormalities.</p

Survival, vitrified two-cell embryo and live offspring rates from either fresh or vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05).</p

Histology and Hematoxylin and Eosin staining of teratoma tissue from ViO-ES9 cells showing differentiation into tissues indicative of the three germ layers (A) including secretory epithelium (i), articular cartilage (ii) and keratinized epithelium (iii).

<p>Immunoflorescent analysis of differentiation into all three germ layers (B): AFP (i); GATA-4 (ii); and NESTIN (iii). Secondary antibodies were labelled with Alexa Fluro® 488 (green) except for GATA-4 which was labelled with Alexa Fluro® 594 (red). Nuclei are stained with DAPI (blue). RT-PCR for Flk-1, VE-Cadherin, PECAM, Vimentin and Nestin (C).</p

Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique

One of the technical bottlenecks in producing nonhuman primate models is that current assisted reproductive techniques, such as in vitro culture and frozen conservation of multicell-stage embryos, often result in poor embryo quality and subsequently lead to low birth rates. We investigated whether pronuclear embryo transfer can be used as an effective means for improving pregnancy and live birth rates of nonhuman primates. We collected 174 metaphase II oocytes by laparoscopy from 22 superovulated mature females and then fertilized these eggs using either in vitro fertilization or intracytoplasmic sperm injection, resulting in a 33.3% and a 50% fertilization rate, respectively. These 66 fertilized pronuclear-stage embryos were then tubally transferred to 30 recipients and led to 7 births and 1 abortion. Importantly, we observed that the highest live birth rate of ≈64% was obtained when the transfer of pronuclear embryos was performed in the presence of new corpus luteum in the ovary of recipients between 24 h and 36 h after estradiol peak. Therefore, our experiments demonstrate that by matching the critical time window in the recipient's reproductive cycle for achieving optimal embryo-uterine synchrony, pronuclear embryo transfer technology can significantly improve the pregnancy rate and live birth of healthy baby monkeys. This efficient method should be valuable to the systematic efforts in construction of various transgenic primate disease models