The Connection Between MHC Relatedness and Mate Preference in Female Lab Rats

The Role of MHC Relatedness

In Female Lab Mouse (M. musculus) Mate Preference

Introduction

Mate choice has been in the consciousness of biological studies since Darwin first proposed its function in the selection and evolution of secondary sexual traits. Females, more often the so called ‘choosey’ sex in most species due to their need to devout a large amount of resources to having progeny, thus are responsible for selecting specific characteristics in males (Jennions & Petrie, 1996). What characteristics they are, how to measure the differences between individuals, and what directional preferences are shown are the questions of interest, including in this study.

Although behavioral and physical characteristics serve as the obvious forms of sexual communication, insights into chemical signals have led to the analyses of pheromones and other indicators of genetic fitness as playing an important role. Phermones not only play roles in mate recognition, but assessment as well. For example, many cockroach species emit a pheromone which signals their place in the dominance hierarchy as well as overall quality (Johansson & Jones, 2007).

Delving further into genetics, we see the major histocompatibility complex (MHC) which is a DNA region central to, among other functions, mating preferences and the immune response. Genetic diversity of this region has been maintained as most species show preference for mating with individuals that have dissimilar MHC genes. This includes mice (Mus musculus) who learn their parents’ odors and then avoid mating with similar-smelling individuals, as the odor is linked to MHC structure (Penn & Ilmonen, 2005).

Laboratory mice are excellent model species for studying mate choice as they have a propensity for mating, grow quickly, communicate primarily through olfaction (both glands and urine), and have had their entire genome sequenced. In this experiment a classical Y-maze mate choice experiment was run in order to examine what male scent female mice preferred, an individual with a more similar or more dissimilar MHC. It was hypothesized that individuals would spend a larger amount of time per trial in the arm which contained the scent of a more MHC dissimilar male.

Methods

Wild house mice caught in Austria were used as the test subjects, specifically as a sample which began with 50 adult females and 100 adult males at the start of the experiment. At the time of testing the average age for both the females and males was 5.2 months. Each pair of males used in the individual Y-maze tests were full-brothers who differed in MHC genotype and were unrelated to the tested female. MHC regions for all individuals was determined via single-strand conformation polymorphism, and then the PCR products were directly sequences to determine alleles. The MHC differences were then calculated using genetic distances at antigen binding sites (Tamura et al., 2007). Scent marks were gathered by placing filter papers in the males’ home cages and collected after around 19 hours on average, when sufficient urine as well as trace saliva and feces had been deposited. Additionally, stimulus paper containing a mixture of female urine was placed into the male home cages, as this increases male marking behavior. The soiled male filter paper was also analyses for number of marking spots and rated on a subsequent scale for future reference.

A Y-maze preference test was utilized to check female odor preference. Females were only tested when they were undergoing estrus, which was induced in some cases by placing previously soiled male bedding in their respective home cages, an observation previously reported (Flowerdew, 1987).

The Y-maze apparatus itself is adopted from Yamazaki et al. (1979), an acrylic chamber divided into a partitioned off start zone at the central tip, followed by a neutral zone, and then breaking off into two equal length arms which served as the choice zones. The measurements were as follows, respectively: 6 x 14 x 6 cm, 6 x 24 x 6 cm, and 6 x 8 x 6 cm. In the center of the choice zone branches there were inserted squares of wire mesh upon which the scent marked filter paper was affixed in front of it. Two fans were placed at the ends of the chambers in order to blow the scents along the length of the apparatus and get the attention of the female test mouse.

A positive control was done using blank filter paper to see if this had any particular attraction for the female mice. Testing began with a 5 minute habituation period for the female mouse in the starting area with no filter paper in place. The partition blocking in the starting zone was then removed and the female mouse was allowed to explore the maze while being recorded. Each female was recorded for 15 minutes before the trial was ended and she was removed from the maze. Furthermore, each trial consisted of different marked filter paper, each respectively belonging to a different brother of a pair, and a different female mouse.

Prior to the final analyses a number of trials were excluded for a variety of reasons, including the female not leaving the starting chamber, differences in testing circumstances, problems with recording length, or inability to match genotypes. In addition, only the first 10 minutes of footage was considered for inspection. A total of 11 videos were looked at in the finalized data section. Video analysis data was measured in seconds of time that the female spent in each of the choice zones of the Y-maze respectively using video software and stop watches. In this case, the female mouse had to have had at least half of her body over the line designating the choice zone to be considered as being in that zone. MHC genetic antigen distance was measured compared to the females as described above earlier in the methods.

Results

Overall, the female mice showed minimal level of preference for MHC dissimilar male scent marks (M = 207.65, SD = 137.5) compared to MHC similar male scent marks (M = 114.33, SD = 70.02). According to spearman’s correlation, MHC dissimilar has an r of -.16 and MHC similar has one of -.22. It may seem surprisingly that both are negative but again they are only very slightly negative and in addition it is likely due to the small range in MHC distances. Regardless, the MHC dissimilar group still shows a slightly greater correlation at least in comparison. Regarding the positive control, female mice show a definite preference for marked filter paper as opposed to blank paper (Figure 1).

Fig. 1 Positive Control for Scented vs Blank Filter Paper Preference

As there are only 11 paired data points in this experiment I elected to go with the non-parametric route when analyzing the mate preference. The Wilcoxon Signed Ranked test revealed that the time spent with dissimilar MHC was not significantly greater than similar MHC (Z = -1.6, p = .110). The power analysis yielded a result of 0.8.

Discussion

Although a slightly greater time was spent overall in the branch where the dissimilar MHC scent was, meaning there was no arbitrary branch preference, the Wilcoxon results suggest no overall MHC preference in this experiment. This is in stark contrast to a large portion of existing research which suggests MHC-dependent mating preferences in female mice is quite strong, usually with an avoidance of MHC-similar males (Penn & Fischer, 2004). It has been hypothesized that this strategy ensures the maintenance of genome-wide MHC diversity (Jordan & Bruford, 1998), meaning immunological variety is also maintained. In more specific terms, it is possible that this mechanism prevents inbreeding depression.

However, this study suggests the opposite, or at least minimizes MHC preference. There are a number of possible explanations. When not in estrus female M. musculus actually show little if any mate preference at all (Zinack & Lima, 2013) and in this case estrus was actually triggered unnaturally so it’s possible this results in differences in mating compared to the natural estrus cycling. Another question in the field is whether MHC preference is absolute or relative, in other words, are there other traits taken into account and are they also given the same weight?

Another important addendum is that although MHC discrimination has been fairly well established for inbred laboratory mouse lines, the same cannot be said for wild mice. One study found that wild mice, even when fostered into inbred lab strains showed little to no MHC-based mating preferences (Eklund, 1999).

Potential limitations of this experiments may also play a role in the results, and if not, should still be taken into account. The sample size was fairly small, and errors when analyzing the video footage are fairly easy to make. Individual differences in the female mice may also play a role, particular if they were hungry or agitated or in some other affective state at the time of testing. Lastly, the urine samples left on the filter paper by the males may not have been equal for both brothers.

Future studies could investigate male MHC-mate choice, as well as varying similar MHC-distance brothers but varying other variables that may be desirable for selection by the female, such as size or other measures of fitness. Overall, we need to know what other measures of fitness are communicated through olfaction and what weight they have in mate choice.