How do Fbxo7 and PI31 control sperm morphogenesis and male fertility?

Sperm cells are the smallest cells in the body, having shed virtually all of their cellular contents in order to swim faster. The production of mature sperm thus involves perhaps the most radical changes in cellular shape in all of mammalian biology. As sperm cells develop, they are dragged into deep indentations ("crypts") within nurse cells (Sertoli cells) that support and sculpt them. Therein, developing sperm cells make ready their cytoplasm for elimination, and package it up for disposal by the Sertoli cells. As the sperm realise their final shape, they are pushed back out of the crypts, and finally released as free-living, mature sperm cells. As yet, little is understood about the processes that coordinate this transformation and control how each cell is moulded into its final shape. We recently discovered that a protein called Fbxo7 is critical for sperm remodelling at a precise time in the process of assuming this streamlined shape. In male mice that lack Fbxo7, sperm cells develop normally up to the point where they enter the crypts - however the cells never leave the crypts and instead all die at once. This shows that Fbxo7 is necessary for the cellular remodelling steps to make a sperm. We know that Fbxo7 plays a role in maintaining the function of mitochondria - which supply cells with energy - and in disposing of defective mitochondria. Fbxo7 performs many of its functions by adding a small tag, called ubiquitin, onto other "target" proteins. This ubiquitin tag usually causes the target proteins to be disposed of by a large degrading enzyme called the proteasome, but in some cases, it can alter the target protein's function or move it around the cell. Fbxo7 also interacts with a partner protein called PI31 that attaches proteasomes to motor proteins to move them around within a cell. Our findings suggest that the addition of ubiquitin to target proteins by Fbxo7, with or without the help of its partner PI31, is likely to be akey factor in regulating mitochondria and/or proteasome trafficking inside the cell during the final phase of sperm remodelling.

Our key goals are:
1. To find out what is going wrong in the testes of males lacking Fbxo7 and PI31 - are the mitochondria fragmented or deformed, are they being carried to the right location within the cell, and are they working normally? Alternatively, is there a problem with proteasome localisation? We will investigate this using fluorescence microscopy and electron microscopy in conjunction with staining for markers of each of these processes.
2. To identify what Fbxo7 is doing biochemically in normal testes - what is it attaching ubiquitin to, and what effect does this have? Does it require help from PI31? What proteins associate with proteasomes when sperms are being sculpted and how is this controlled by Fbxo7 and PI31. We will investigate this by comparing the ubiquitin-tagged proteins and the proteasome-associated proteins in cells from mice lacking Fbxo7 and PI31.

Understanding how these events take place is important from both a pure science and a medical perspective - for example, understanding Fbxo7 function may help us understand and eventually treat sterility in some infertile patients who make no sperm. Conversely, selectively interfering with Fbxo7 function in the testes could become the basis for novel methods of male contraception. Fbxo7 is also important in other tissues: many different cell types such as red blood cells and nerve cells also undergo remodelling to transform their shapes, and Fbxo7 deficiency is also associated with anaemia and neurological conditions such as Parkinson's disease. Understanding how Fbxo7 and PI31 control cell shape in the testis may shed light on their role(s) in these other diseases as well.

Cellular remodelling is critical for the production of functional sperm. While there is extensive descriptive information available on how germ cells and Sertoli cells cooperate to dispose of excess germ cell cytoplasm and sculpt the mature sperm cell shape, little is thus far understood about the underpinning biochemistry and the pathways involved. We have identified a novel mouse that specifically perturbs the final stages of sperm differentiation. Male mice homozygous for a hypomorphic mutation in Fbxo7 are sterile, and developing spermatids are phagocytosed at the point they would normally start to remodel their cytoplasm. Fbxo7 is the substrate recognition component of an SCF-type E3 ligase complex, implicating ubiquitination as a key factor in late stages of sperm development. Fbxo7 interacts with PI31, which we have recently described as an adaptor for the SCF-Fbxo7 ubiquitin ligase, and which has also been described as a proteasome transporter in other cell types. Evidence from related fruit fly and mouse models, together with Fbxo7 and PI31's known roles in mitophagy and proteasome regulation, highlight these processes as being of particular interest for investigation. This project will build on our previous work by combining cutting edge super-resolution and electron microscopic investigation of sperm ultrastructure with a proteomics-based investigation of the disrupted ubiquitination pathways and proteasome interactiomes in Fbxo7 and PI31 mutant testes. These studies will bridge the gap from genotype to phenotype and trace how defective Fbxo7 ubiquitination affects mitochondrial remodelling, proteasome localisation and autophagic / phagocytic activity during sperm development. Concurrent with this, we will explore dosage effects and the contribution of testes cell lineages by comparing a targeted deletions of Fbxo7 and PI31 to the existing Fbxo7 hypomorphic mutant.