Effects of ooplasm manipulation on DNA methylation and growth of progeny in mice

Abstract

New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples in which natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on postnatal growth and development using a mouse genetic model, with particular emphasis on the possible effects of intergenotype manipulations. We performed interstrain and control intrastrain maternal pronuclear transfers, metaphase-II spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice, and found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter- and intrastrain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intrastrain germinal vesicle transfers (GVTs). Interstrain GVT females, but not males, had significantly lower body weights at birth and thereafter compared with the intrastrain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, while intergenotype GVT can result in significant growth deficiencies after birth.

FIG. 1.

Maternal PNT mice. A) Illustration of mPNTs. C57BL/6 (B6) components are black/gray and DBA/2 (D2) are white, with mPNs and pPNs shown as small and large circles, respectively. Resulting combinations from mPNTs are below. B) Postnatal growth of mPNT females. Body weights are compared between interstrain transfers BDB (n = 16), DBD (n = 20), intrastrain transfers DDB (n = 21), BBD (n = 28), and non-PN transfers DB (n = 18) and BD (n = 16). C) Postnatal growth of mPNT males. Body weights are compared between interstrain transfers BDB (n = 18), DBD (n = 14), intrastrain transfers DDB (n = 29), BBD (n = 29), DB (n = 11), and BD (n = 14).

FIG. 2.

ST mice. A) Illustration of STs. C57BL/6 (B6) components are black/gray and DBA/2 (D2) are white, with maternal spindles shown as large ovals and sperm as small ovals. Resulting combinations from STs and ICSI are below. B) Postnatal growth of ST females. Body weights are compared between interstrain ST-DBD (n = 22) and controls ST-DDB (n = 18), ICSI-BD (n = 17), and ICSI-DB (n = 17). C) Postnatal growth of ST males. Body weights are compared between interstrain ST-DBD (n = 17) and controls ST-DDB (n = 21), ICSI-BD (n = 8), and ICSI-DB (n = 8).

FIG. 3.

CT mice. A) Illustration of CTs. C57BL/6 (B6) components are black/gray and DBA/2 (D2) components are white, with maternal spindles as large ovals, sperm as small ovals, and transferred cytoplasm as small circles. Note that cytoplasm is also injected with the sperm. Resulting combinations from CTs and ICSI are below. B) Postnatal growth of CT females. Body weights are compared between interstrain CT-DBDB (n = 13) and controls CT-DDDB (n = 17) and ICSI-DB (n = 17). C) Postnatal growth of CT males. Body weights are compared between interstrain CT-DBDB (n = 15) and controls CT-DDDB (n = 9) and ICSI-DB (n = 8).

FIG. 4.

GVT mice. A) Illustration of GVTs. C57BL/6 (B6) components are black/gray and DBA/2 (D2) are white, with GV nuclei shown as circles, MII nuclei as ovals, and sperm as diamonds. Resulting combinations from GVTs and ICSI are below. B) Postnatal growth of GVT females. Body weights are compared between interstrain GVT-DBD (n = 17) and controls GVT-BBD (n = 12), IVM-BD (n = 15), and IVM-DB (n = 16). Interstrain GVT-DBD females are smaller than intrastrain GVT-BBD and IVM-BD from 3 to 13 wk (“a” denotes P < 0.03; “b” denotes P < 0.006). C) Postnatal growth of GVT males. Body weights are compared between interstrain GVT-DBD (n = 16) and controls GVT-BBD (n = 16), IVM-BD (n = 21), and IVM-DB (n = 19). All embryos were fertilized in vitro by ICSI.