Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility

Abstract

The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.

Figure 1

Targeting strategy. (A) Structure of the mouse Afp genomic locus with the representation of the two promoters (P1 and P2) and the targeting vectors pAFP KO1 (pKO1) and pAFP KO2 (pKO2). The Afp exons are shown as dashed boxes. The Afp flanking probe used for Southern blotting is represented (black box). The EcoRI (E) site used to detect the polymorphism generated by the homologous recombination of pAFPKO1 and pAFPKO2 is indicated. LacZ indicates the IRES-LacZ cassette used as reporter, and Neo is the Pgk Neo selectable marker used to select for insertion of the vectors. The LoxP sites flanking the Neo cassette in pAFPKO2 are indicated. pBS represents the plasmidic pBSIIKS+ vector (Stratagene); tk2 is the thymidine kinase marker used to select for homologous recombination. (B) Example of Southern blot analysis of genomic DNA of wild-type, heterozygous (Afp^tm1Ibmm/+), and mutant Afp^{tm1Ibmm/tm1Ibmm} animals: the arrows point to the 9.5-kb wild-type (wt) and the 6.5-kb mutant (mt) bands.

Figure 2

(A) Northern blot analysis of intercross litters. Total RNA from 16.5 E embryos (genotyped by Southern blot) was analyzed by Northern blot. The mouse Afp probe detects a 2.2-kb transcript. A human GAPDH probe was used as loading control. (B) Western blot analysis with protein extracts from different tissues. Pure AFP, albumin (ALB), and adult liver (A. liver) were used as controls. Extracts were from wild-type (+/+), heterozygous (+/−), and Afp^{tm1Ibmm/tm1Ibmm} mutant (−/−) mice. Protein from fetal liver (F. Liver), yolk sac, and amniotic fluid were tested with a serum raised against AFP (Ab-AFP). The same blot was tested with a serum raised against albumin (Ab-ALB).

Figure 3

Single embryo implantations. (A) Heterozygous Afp^tm1Ibmm/+ females and homozygous Afp^{tm1Ibmm/tm1Ibmm} mutant males were mated. Blastocysts were collected and implanted individually into pseudopregnant females. (B) Southern blot analysis of mice obtained from different females.

Figure 4

Quantitative RT-PCR on the different genes of the albumin family. Embryos (15.5 E) from intercrosses matings were dissected and genotyped, and total RNA from their livers was extracted. The concentration of Gapdh transcript was measured for each sample tested, and the ratio of the tested transcript [Afp, albumin (Alb), alpha-albumin (Alf)] on the Gapdh transcript was calculated. The relative amounts the three mRNAs tested is given for wild-type embryos (Afp+/+), heterozygous (Afp+/−), or homozygous (Afp−/−). The number of each sample tested for each genotype is indicated in parentheses.

Figure 5

Anatomical and histological analysis of Afp mutant (Afp^{tm1Ibmm/tm1Ibmm}) ovaries and uteri of preburtal (week 4) and adult (week 12) mice. (1A) Uterine horn and ovary of a 4-week-old Afp^{tm1Ibmm/tm1Ibmm} mutant female. (1B) Uterine horn and ovary of a wild-type 4-week-old female. (2A) Uterine horn and ovary (arrowhead) of an adult Afp^{tm1Ibmm/tm1Ibmm} mutant female. (2B) Ovary from a 12-week-old Afp^{tm1Ibmm/tm1Ibmm} mutant female. (2C) Ovary from a 12-week-old wild-type female. The surface distortions caused by large corpora lutea are not observed in the Afp^{tm1Ibmm/tm1Ibmm} mutant female. (2D) General histological structure of an Afp^{tm1Ibmm/tm1Ibmm} mutant ovary (section from a 16-week-old female) showing that mature Graafian follicles (*) are present. (2E) At the same age, wild-type ovaries exhibit large corpora lutea (§), indicative of successful ovulation (these structures were never found in Afp^{tm1Ibmm/tm1Ibmm} mutant ovaries). (Magnifications: 1A and 2A, ×2.5; 2B and 2C, ×10; and 2D and 2E, ×25.)

Figure 6

Hormonal levels. Each point corresponds to a single mouse. (Upper) Results of progesterone and estradiol assays in Afp^{tm1Ibmm/tm1Ibmm} mutant females and controls. Assays were performed on serum from different batches of females maintained for at least 6 weeks in three different cages (A, B, C). Note the lack of progesterone in the mutant mice; the difference with the control mice is significant (P = 0.05). (Lower) Results of gonadotropin (LH and FSH) measurements in wild-type and Afp^{tm1Ibmm/tm1Ibmm} mutant females ovarectomized (OV: first two series) or not ovarectomized (last two series). The difference in the LH levels is significant (P = 0.01), whereas that in the FSH levels is not (P = 0.16).