This post is by Kimberly Davenport, first year graduate student in Animal Science with Dr. Brenda Murdoch at the University of Idaho and Brenda Murdoch, assistant professor of animal genetics at the University of Idaho.
Kim Davenport
With each research project comes its own challenges, rewards, and quirks. My research project in Dr. Brenda Murdoch’s laboratory involves characterizing homologous recombination in different mammalian species. Homologous recombination is an extremely important process in gametogenesis that not only contributes to genetic variation but also ensures proper chromosome segregation during cell division in meiosis I. Errors due to failure or improper placement of recombination represent a significant contribution to aneuploidy, developmental disabilities, fetal loss and infertility [1]. Despite the importance, we know very little about the factors that control and/or influence global meiotic recombination in mammals.
Work in model organisms; yeast, fruit flies, and mice, have outlined the steps in the recombination pathway [2]. For example, we have gained insights on the sequential relationship of chromosome pairing, sister chromatid cohesion, synapsis and recombination. Meiotic recombination is initiated by a topoisomerase like protein (SPO11) that establishes breaks in the chromosomes. Chromosome breaks are resected with the aid of strand invasion proteins RAD51 and DMC1 and ultimately produce double Holliday junction intermediates. These are resolved as either crossovers or non-crossovers. The majority of non-crossovers are generated early in the pathway and only a subset of the breaks are resolved as crossovers. Crossover repair protein MLH1 localizes the majority of crossovers [3]. Interestingly, only a few of the breaks are resolved as crossovers and the rules that govern the frequency and sites remain unclear.
Over many years of research, scientists have identified a few general guidelines associated with meiotic recombination. First, one recombination event, or crossover, is required per chromosome arm pair for proper cell division. Second, we know from previous studies that the placements of recombination events are not random. Crossovers exhibit preferences in the genome called “hotspots” and experience “interference” in that one crossover cannot occur too close in proximity to another [4]. Third, crossover numbers have been shown to be sexually dimorphic (different between males and females) in many different species. And lastly, the number of crossovers is correlated with the size of the genome and number of chromosome arms (i.e. the more chromosome arms, the more crossovers are likely to occur).
Although recombination occurs in many different species, our lab focuses primarily on livestock species, specifically sheep and cattle. These two domestic species are not only important for agricultural food production, but also provides an interesting biological comparison and insight into how recombination has evolved in ruminants. While sheep and cattle have the same number of chromosome arms and a similar genome length, they do not have the same number of genome-wide recombination events. This poses a number of important questions about how genomic meiotic recombination levels are controlled in different domesticated species.
In the Murdoch lab we identify and characterize crossover events using our optimized immunohistochemistry method. This cytogenetic approach allows us to directly observe where chromosomes recombine through the microscope. We identify crossovers with an immunofluorescent antibody which specifically binds to MLH1, a protein that is known to be involved in repairing DNA breaks into crossovers.
Figure 1: Meiotic cross-overs in sheep and cattle. A) Representative images of MLH1 foci on synatonemal complex from a Gelbvieh bull (46 MLH1 foci), and B) Targhee ram (71 MLH1 foci) spermatocyte.
So, how do we get samples to collect the data we need? Much like a surgeon is on call for patients who need assistance, I serve as the “on call” graduate student for samples! Since we study primary spermatocytes, we require testicular samples from males who have reached puberty or older. However, finding males that have not been castrated provides a real challenge. Male sheep and cattle are usually castrated early in life to provide a safer working environment for both the animals and the people who care for them. Some males are kept intact (not castrated) for breeding purposes, but these are the few “best” animals. So, any time an intact male is at the end of his breeding career and is harvested for food, I retrieve a testicular sample. But there is one more problem: these samples cannot be frozen, and we need to extract the cells and fix them on microscope slides within 24 hours. Otherwise, the integrity of protein we use to identify crossovers degrades.
Prepping these samples is a race against time. If we receive the samples from a local source, I pick them up as soon as I get a phone call. If they are shipped overnight from elsewhere, I wait impatiently for the samples to arrive the next day. In a way, I am considered “on call” for receiving and prepping our samples. And since these samples only last for about 24 hours on ice, I prep them almost immediately upon arrival. It is exciting in our lab to receive a new sample because the data we collect from one ram or bull may be very different from another!
The reward for timely but precise preps of our samples is that we are able to learn more about the process of meiotic recombination, and impact the way the scientific community understands how genes are passed from generation to generation. Since meiotic recombination contributes to genetic variation, understanding differences as well as the mechanism behind the process gives us deeper insight into how genetic diversity is evolving.
References:
- Hassold, T., and Hunt, P. (2001). To err (meiotically) is human: the genesis of human aneuploidy. Nature reviews. Genetics 2, 280-291.
- Murdoch, B., Owen, N., Stevense, M., Smith, H., Nagaoka, S., Hassold, T., McKay, M., Xu, H., Fu, J., Revenkova, E., et al. (2013). Altered cohesin gene dosage affects Mammalian meiotic chromosome structure and behavior. PLoS genetics 9, e1003241.
- Baudat F and de Massy B (2013). Meiotic recombination in mammals: localization and regulation. Nature Reviews Genetics 14, 794-806.
- Jones, G.H., and Franklin, F.C. (2006). Meiotic crossing-over: obligation and interference. Cell 126, 246-248.
