Testicular Leydig cells produce androgens essential for proper male reproductive development and fertility. Here, we describe a new Leydig cell ablation model based on Cre/Lox recombination of mouse Gata4 and Gata6, two genes implicated in the transcriptional regulation of steroidogenesis. The testicular interstitium of adult Gata4flox/flox;Gata6flox/flox mice was injected with adenoviral vectors encoding Cre + GFP (Ad-Cre-IRES-GFP) or GFP alone (Ad-GFP). The vectors efficiently and selectively transduced Leydig cells, as evidenced by GFP reporter expression. Three days after Ad-Cre-IRES-GFP injection, expression of androgen biosynthetic genes (Hsd3b1, Cyp17a1, Hsd17b3) was reduced, whereas expression of another Leydig cell marker, Insl3, was unchanged. Six days after Ad-Cre-IRES-GFP treatment, the testicular interstitium was devoid of Leydig cells, and there was a concomitant loss of all Leydig cell markers. Chromatin condensation, nuclear fragmentation, mitochondrial swelling, and other ultrastructural changes were evident in the degenerating Leydig cells. Liquid chromatography-tandem mass spectrometry demonstrated reduced levels of androstenedione and testosterone in testes from mice injected with Ad-Cre-IRES-GFP. Late effects of treatment included testicular atrophy, infertility, and the accumulation of lymphoid cells in the testicular interstitium. We conclude that adenoviral-mediated gene delivery is an expeditious way to probe Leydig cell function in vivo. Our findings reinforce the notion that GATA factors are key regulators of steroidogenesis and testicular somatic cell survival.
The microtubule‐based cilium (also known as a flagellum) extends from the surface of most eukaryotic cells. This long, slender organelle is biochemically complex and is conserved in many eukaryotic organisms. The unicellular alga, Chlamydomonas reinhardtii, serves as a tractable organism for studying the assembly and function of cilia. Cilia provide locomotion and environmental sensing. To produce motility, specialised molecular motors called outer and inner dynein arms generate force to produce waveform. These motors are regulated by other macromolecular complexes in the cilium. The ease of genetic and biochemical approaches combined with electron microscopy has provided the ability to perform in‐depth studies of the regulation of ciliary assembly and function. Results from Chlamydomonas researchers have broadened our knowledge of motile cilia diseases in humans. There are still many outstanding questions to be addressed. Key Concepts Chlamydomonas reinhardtii is a haploid, motile, single‐celled alga that is useful for the study of cilia. The Chlamydomonas cilium is a complex structure that generates asymmetric and symmetric waveforms to allow forward and backward swimming, respectively, and orientation to environmental signals. During ciliary motility, dynein arms pull microtubule doublets past each other to generate sliding, which is converted into axonemal bending, although the mechanism for how this occurs is still controversial. Outer dynein arms control the ciliary beat frequency by changing the velocity of doublet sliding. Inner dynein arms in conjunction with the radial spokes and the central pair apparatus, control the bend amplitude and the shape of the waveform. The axoneme is divided into 96 nm repeating segments that contain regulatory, enzymatic and structural components essential for motility; these include the inner and outer dynein arms, N‐DRC, MIA complex and radial spokes as well as two central microtubules. IFT (intraflagellar transport) is essential for building the cilium by transporting cargo into (anterograde) and out of (retrograde) the cilium. Cell biological research about ciliary components, including the central pair apparatus, radial spokes and N‐DRC, provides key information about how ciliary motility is regulated. Genetic, biochemical and microscopic methods used in Chlamydomonas have allowed molecular resolution of interactions between regulatory complexes. Studies of Chlamydomonas cilia have provided additional insights to the structure, function and regulation of cilia, and have helped with the understanding of human cilia‐related diseases called ciliopathies.
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