We created P450c17 gene-knockout mice to study the function of DHEA and DHEAS in the developing rodent nervous system. However, we unexpectedly found that P450c17 is expressed and is catalytically active in the early embryonic endoderm, a site not previously known to express steroidogenic enzymes, and we found that mice lacking P450c17 die by E7. The embryonic lethality was not due to inadvertent deletion of another gene overlapping the P450c17 locus. Analysis of the mouse genome database indicates that no other genes are located within at least 500 kb of the mouse P450c17 gene.
Steroid hormones are essential for reproduction and for the maintenance of pregnancy, but the sources and roles of these hormones vary dramatically among various mammalian species. Estrogen and progesterone are necessary to establish and sustain pregnancy in mammals (1
). Steroid synthesis in pregnancy involves the maternal ovarian corpus luteum, the placenta, and, in some species, the fetus as well. Progesterone is required to suppress uterine contractility and maintain pregnancy (20
). In human beings, a luteal-placental shift occurs by the end of the first trimester, and the maternal ovary is no longer necessary after this time (42
). In rabbits, no fetal or placental steroidogenesis is needed, as spontaneously occurring knockouts of the gene for P450scc develop normally, survive to term, and die shortly thereafter from adrenal insufficiency (37
). In rodents, both maternal ovarian and fetal placental steroidogenesis were thought to be necessary throughout pregnancy (reviewed in reference 8
). However, mouse fetal steroidogenesis appears not to be necessary, as P450scc−/−
-knockout mice survive to term like the rabbit and die shortly thereafter from adrenal insufficiency (24
). We also found that P450scc is expressed in the embryo at E7, in the same region as P450c17 (data not shown). Therefore, if the mouse embryo normally expresses P450scc, and if embryonic and placental P450scc activities are indeed necessary for maintenance of pregnancy, it is possible that P450scc−/−
mice might be able to survive by using pregnenolone provided by the mother as the precursor for further steroid hormone production. P450c17 is expressed in several layers of the developing rodent placenta (47
), although it is not expressed in the human placenta (6
). However, the rodent placenta does not produce C19
steroids before day 11 due to the lack of P450c17 mRNA, protein, and enzyme activity (19
). The P450c17-knockout mice die 4 days before this, at a time when the embryo has implanted but may not yet be placentally dependent. Thus, it is unlikely that placental failure due to lack of steroidogenesis is the cause of fetal demise in P450c17-knockout mice.
In addition to placental expression of steroidogenic enzymes, the mouse uterine decidua also expresses P450scc and 3β-hydroxysteroid dehydrogenase type VI and produces progesterone (2
). These data suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy and that local production of progesterone may act as a local immunosuppressant, preventing rejection of the fetus. However, while the decidua may produce progesterone, it is a maternal tissue that would not be affected in the P450c17+/−
mothers, and no role for decidual steroids produced by P450c17 has been shown. Indirect evidence suggests that the rabbit, rat, and mouse blastocyst must produce estradiol in order to implant (16
). If so, this could explain the embryonic lethality in P450c17−/−
mice. However, this mechanism is not generally true for other mammals, and it is not compatible with the survival of P450aro−/−
mice to term. Human beings with homozygous defects that destroy all P450c17 protein activity or P450c17 gene expression develop normally and survive to adulthood, having a phenotype of sexual infantilism; sex reversal in 46, XY genetic males; and relatively mild disorders of adrenal steroid production (reviewed in reference 3
). Thus, the lethal phenotype of P450c17−/−
mice is unexpected and unprecedented, vividly highlighting the profoundly different strategies employed by different mammalian orders in handling the endocrinology of pregnancy and development.
The steroidogenic activity of P450c17 requires two proteins, P450c17 and its obligatory electron transfer partner cytochrome P450 reductase (Cpr
mice are also embryonic lethal but not until E9.5 (35
), well after the death of P450c17−/−
mice. These observations suggest that P450c17 may have another, heretofore unanticipated activity, other than as a P450 enzyme.
The events occurring in the embryo following implantation and prior to gastrulation are now being established. After implantation, the embryo begins to generate an anteroposterior axis. This axis may be formed using cues from the extraembryonic tissues or from the mother. While the location of the primitive streak and subsequent gastrulation of the embryo is crucial for the orientation of both the anteroposterior and dorsoventral axes, signals from the embryonic endoderm prior to gastrulation have been shown to be important for initiation of the anteroposterior axis (reviewed in references 6
). These signals may be initiated by several transcription factors such as GATA4
β, and Hesx1
. Other factors expressed in the embryonic endoderm at ~E6 include goosecoid
, and transforming growth factor β. GATA4−/−
, and nodal embryos fail to gastrulate and die at about E6 to E7. As P450c17 is expressed and is enzymatically active in the embryonic endoderm about this time, its potential steroid products may be additional factors that play a role in the cell movement in the embryonic endoderm and in the generation of the anteroposterior axis of the embryo.