The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive

Pluripotence in embryonic stem (ES) cells and the early mouse embryo is controlled by several key transcription factors such as Oct4, Sox2, Foxd3, Nanog, and signaling molecules such as STAT3, FGF4, FGF4 receptor, and LIF (4,11,18,22,32,35,51). The regulation of expression of these key factors is thus crucial for the maintenance of pluripotence and embryonic development. However, little is known about the upstream factors that regulate the expression of these genes. Oct4, a member of the POU homeodomain family of transcription factors, is the best studied of these factors. Recent studies have shown that Oct4 plays a critical role in embryonic development and cellular differentiation (38,43). Oct4 is expressed in totipotent and pluripotent stem cells of the pregastrulation embryo, primordial germ cells, and oocytes (37,44,47). Oct4 is also highly expressed in ES and embryonic carcinoma cell lines, such as P19 and F9 cells, and is rapidly down-regulated by differentiation induced with retinoic acid (RA) (6, 31). In ES cells, a less than twofold increase in the level of Oct4 mRNA causes the cells to differentiate into primitive endoderm and mesoderm, whereas reduction to less than 50% of normal levels triggers differentiation into trophectoderm (36). Moreover, targeted disruption of the Oct4 gene in mice results in embryonic death at the blastocyst stage and compacted morula cells that do not differentiate along the pluripotent inner cell mass lineage but instead differentiate into trophectodem (32).

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.

To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage lambda shuttle vectors integrated in the genome in a head-to-tail arrangement. As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector. The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C. The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively. Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 x 10(-5) at 250 mg of the alkylating agent per kilogram of body weight. At this dose, in liver DNA of the same mice, mutation frequencies were approximately 3 x 10(-5). DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G.C----A.T transitions. These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo. In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.