Vitamin D-dependent rickets type I (VDDR-I), also known as pseudovitamin D deficiency rickets (PDDR), is an autosomal recessive disorder characterized by low or undetectable levels of 1␣,25(OH) 2 D, secondary hyperparathyroidism, hypocalcemia, hypophosphatemia, and severe rachitic lesions (18 -21). VDDR-I is assumed to result from impaired synthesis of 1␣,25(OH) 2 D, and, indeed, a number of 1␣(OH)ase gene mutations have been reported in this disorder that result in diminished or absent 1␣(OH)ase activity (13,(22)(23)(24)(25)(26).To further investigate the functional role of the 1␣(OH)ase enzyme, we generated mice deficient in 1␣(OH)ase by gene targeting. Materials and MethodsMethods including construction of the 1␣(OH)ase targeting vector; transfection of embryonic stem (ES) cells and generation of 1␣(OH)ase-deficient mice; Southern blot and PCR analysis of ES cell and mouse tail DNA; Northern blot analysis; biochemical and hormonal analyses; histological analysis; computer-assisted image analysis; immunohistochemistry; and f luorescenceactivated cell sorter (FACS) lymphocyte phenotyping are presented in the supplemental data (which is published on the PNAS web site, www.pnas.org). ResultsThe targeting vector shown in Fig. 1A was used to inactivate one allele of the 1␣(OH)ase gene in ES cells. The inactivated allele lacked both the hormone-binding domain and the heme-binding domain of the enzyme. Two independent ES cell clones were used to generate two lines of mice heterozygous for the mutation, which were then interbred to generate 1␣(OH)ase null (Ϫ͞Ϫ) mice (Fig. 1B). Litter sizes were no different from normal, and the mutated allele was transmitted to the progeny with the expected Mendelian frequency. Thus, haploinsufficiency of the 1␣(OH)ase did not affect embryonic survival. By reverse transcription (RT)-PCR, renal expression of the kidney 1␣(OH)ase mRNA in (ϩ͞Ϫ) mice was reduced relative to that in (ϩ͞ϩ) mice, and, in (Ϫ͞Ϫ) mice, it was undetectable (Fig. 1C).Circulating concentrations of 1,25(OH) 2 D were undetectable in the homozygous null mice and were somewhat lower (although not significantly so) in the heterozygotes relative to normals at 7 weeks of age (Table 1). Serum 25(OH)D concentrations were elevated in (Ϫ͞Ϫ) mice relative to the heterozygotes and normals. Both serum calcium and phosphate concentrations were reduced in (Ϫ͞Ϫ) mice relative to the (ϩ͞Ϫ) mice that were normal, and urinary phosphate was increased in the homozygous null mice. Serum parathyroid hormone concentrations were markedly elevated, the alkaline phosphatase concentrations were twice normal, and the body weight was substantially reduced in the homozygous null mice at this time (Table 1). The null mutant mice appeared grossly normal from birth until This paper was submitted directly (Track II) to the PNAS office.Abbreviations: 1␣(OH)ase, 25(OH)D-1␣-hydroxylase; VDDR-I, vitamin D dependent rickets type I; VDR, vitamin D receptor.

ABSTRACT

We have previously shown that rat ovaries synthesize atrial natriuretic peptide (ANP) and express the cognate guanylyl cyclase (GC-A and GC-B) receptors for ANP. Since another natriuretic peptide, termed the C-type natriuretic peptide (CNP), can also interact with these receptors, we have investigated whether rat ovaries express CNP and if so, whether the concentration of this natriuretic peptide and the guanylyl-cyclase receptors are influenced by the estrous cycle. CNP mRNA was detected in rat ovaries using a reverse transcription (RT) polymerase chain reaction (PCR) strategy. RIA of ovarian extracts, obtained at the individual days of the estrous cycle, revealed the presence of immunoreactive CNP. The highest levels of CNP were detected at proestrus and were approximately 4-fold higher than the levels seen at any other stage of the cycle. GC-A and GC-B receptors were detected using quantitative autoradiography after application of either [125I]ANP or [125I]-tyr0CNP to sections of frozen ovaries. The highest specific binding of each radiolabeled ligand was seen in ovaries from proestrous animals. The GC-B receptors were localized to the membrana granulosa of developing ovarian follicles. Using quantitative PCR, we determined that levels of GC-A and GC-B mRNAs were highest in the ovaries of proestrous animals and were approximately 2- to 3-fold higher than the levels seen at diestrus. These findings demonstrate that a natriuretic peptide system, consisting of ligands and receptors, is present in the rat ovary. Since CNP and the GC receptors show coordinate estrous cycle-dependent variation with maximal expression at proestrus, we speculate that the natriuretic peptides may play an important role in either the development of ovulatory follicles or in the ovulatory process.

A chronic anovulatory polycystic ovarian (PCO) condition can be induced in rats with estradiol valerate (EV). We have previously shown that the early stages (8-10 wk after EV treatment) of the condition are characterized by low basal plasma luteinizing hormone

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