Reproductive abnormalities in mice expressing omega-3 fatty acid desaturase in their mammary glands

Abstract

The Caenorhabditis elegans n-3 fatty acid desaturase (Fat-1) acts on a range of 18- and 20-carbon n-6 fatty acid substrates. Transgenic female mice expressing the Fat-1 gene under transcriptional control of the goat β-casein promoter produce milk phospholipids having elevated levels of n-3 polyunsaturated fatty acids (PUFA). However, females from this line were also observed to have impaired reproductive performance characterized by a smaller litter size (2.7 ± 0.6 vs. 7.2 ± 0.7; P < 0.05) than wildtype controls. While there is a close association between PUFA metabolism, prostaglandin biosynthesis, and fertility; reproductive problems in these mice were unanticipated given that the Fat-1 transgene is primarily expressed in the lactating mammary gland. Using multiple approaches it was found that Fat-1 mice have normal ovulation and fertilization rates; however fewer embryos were present in the uterus prior to implantation. Small litter size was also found to be partly attributable to a high incidence of post-implantation fetal resorptions. Embryo transfer experiments revealed that embryos developing from oocytes derived from transgenic ovaries had an increased rate of post-implantation resorption, regardless of the uterine genotype. Ovary transplantation between Fat-1 and C57BL/6 wildtype females revealed that non-ovarian factors also contributed to the smaller litter size phenotype. Finally, surgical removal of the mammary glands from juvenile Fat-1 mice increased the subsequent number of implantation sites per female, but did not lessen the high rate of post-implantation resorptions. In conclusion, we herein report on a system where an exogenous transgene expressed predominately in the mammary gland detrimentally affects female reproduction, suggesting that in certain circumstances the mammary gland may function as an endocrine regulator of reproductive performance.

Fig. 1

Photo of a representative reproductive tract from a pregnant β-casein Fat-1 female. Two normally-developing pups are accompanied by six implantation sites undergoing resorption. Sites undergoing resorption are denoted by arrow heads (►)

Fig. 2

Mean number of implantation sites (±SEM) that were either resorbing (shaded) or contained a viable fetus (unshaded) at 12 days post coitus following embryo transfer. The number of females in each group were WT/WT, n = 6; WT/Fat-1, n = 5; Fat-1/WT, n = 6; Fat-1/Fat-1, n = 5. Bars with different letters are significantly different (P < 0.05). The total number of implantation sites did not significantly differ across all groups

Fig. 3

Mean number of pups born (±SEM) to dams in various ovarian transplant treatment groups after their first gestation. (A) WT sham (n = 7); (B) β-casein Fat-1 recipient of WT ovary (n = 7); (C) WT recipient of β-casein Fat-1 ovary (n = 6); (D) WT non-surgical control (n = 5); (E) β-casein Fat-1 non-surgical control (n = 8). Bars with different letters are significantly different (P < 0.05)

Fig. 4

Mean number of a resorbing fetuses and b normally-developing pups (±SEM) in various ovarian transplant groups at 12 days post coitus during the second post-operative gestation. (A) WT sham (n = 7); (B) β-casein Fat-1 recipient of WT ovary (n = 7); (C) WT recipient of β-casein Fat-1 ovary (n = 6); (D) WT non-surgical control (n = 5); (E) β-casein Fat-1 non-surgical control (n = 8). Bars with different letters are significantly different from each other (P < 0.05)

Fig. 5

Mean number of implantation sites (±SEM) at 12 days post coitus following mastectomy in β-casein Fat-1 (n = 11), WT (n = 10), and non-mastectomized β-casein Fat-1 females (n = 19). White bars represent developing pups; grey shading represents fetal resorptions. Bars with different letters are significantly different from each other (P < 0.05)

Fig. 6

RT-PCR analysis of mammary tissue from Fat-1 and WT virgin females. The top gel a shows PCR products generated using transgene specific primers. The bottom gel b shows PCR products from primers for mouse β-actin. Lanes 1 and 6: 1 kb Plus Ladder; lane 2: β-casein Fat-1, mammary tissue from 8 week old-virgin female; lane 3: WT 8 week old-virgin mammary tissue; lane 4: β-casein Fat-1 lactating mammary tissue (positive control); Lane 5: H₂O (negative control)

Fig. 7

Milk phospholipid profiles (±SEM) for the substrate and product of the Fat-1 gene for samples derived from WT (white bars; n = 9), and transgenic females expressing the Fat-1 gene under the control of the constitutively active β-actin promoter (Kang et al. ; gray bars; n = 8), or the goat β-casein promoter (black bars; n = 6). Bars with different letters within a fatty acid group are significantly different from each other (P < 0.05)