It is widely accepted that in mammals a causal relationship exists between postcopulatory sexual selection and relative testes mass of the species concerned, but how much it determines sperm size and shape is debatable. Here we detailed for the largest murine rodent tribe, the Rattini, the interspecific differences in relative testes mass and sperm form. We found that residual testes mass correlates with sperm head apical hook length as well as its angle, together with tail length, and that within several lineages a few species have evolved highly divergent sperm morphology with a reduced or absent apical hook and shorter tail. Although most species have a relative testes mass of 1-4%, these derived sperm traits invariably co-occur in species with much smaller relative testes mass. We therefore suggest that high levels of intermale sperm competition maintain a sperm head with a long apical hook and long tail, whereas low levels of intermale sperm competition generally result in divergent sperm heads with a short or non-existent apical hook and shorter tail. We thus conclude that sexual selection is a major selective force in driving sperm head form and tail length in this large tribe of murine rodents.

This study explores the potential effects of interspecific differences in breeding systems on testis organisation and sperm morphology of native murid rodents. It poses the questionwhat are the effects of depressed levels of intermale sperm competition, as indicated by small relative testes mass, on the morphology of testes and spermatozoa in murine rodents? Species from three separate murine tribes, those of the Hydromyini, Rattini and Arvicanthini, were investigated with low relative testes mass being used as a proxy for low, or non-existent, levels of intermale sperm competition. The findings show that in only one of the tribes with low relative testes mass, that of the Hydromyini, was there a reduced testicular area occupied by seminiferous tubules, but in two of the tribes significantly smaller seminiferous tubule diameters were present. In all three tribes, most species with low relative testes mass had a highly derived sperm morphology that, unlike in species with large relative testes mass, had spermatozoa where the head lacked an apical hook, was highly variable in form, often had a very large acrosome overlying the apical region of the nucleus which suggests divergent processes of sperm-egg interaction at the time of fertilisation, and a significantly shorter tail. It is therefore hypothesised that, unlike in species with a large relative testes mass where high levels of intermale sperm competition maintain a streamlined sperm head with an apical hook to aid in zona penetration, in most species of murine rodents with low relative testes mass there is greater reliance on enzymatic digestion of the extracellular matrix around the egg, and especially that of the zona pellucida, to facilitate sperm penetration at the time of fertilisation.

Green Open AccessAll journals published by CSIRO Publishing allow authors to deposit the Accepted version of their manuscript into an institutional repository or put it on a personal website, with no embargo.The Accepted version is the author-created, peer-reviewed, accepted manuscript. The Publisher's edited or typeset versions cannot be used. The institutional repository should be that of the institution employing the author at the time the work was conducted or PubMed Central. We ask that authors link to the published version on the CSIRO Publishing website, wherever possible. The male gamete, the spermatozoon, exhibits considerable interspecies morphological variation 17 across mammals, especially among murid rodents. In Australasia most murids in the Tribe 18Hydromyini have a spermatozoon with a highly complex head that has, in addition to an apical hook 19 characteristic of most murids, two further projections that extend from its upper concave surface, 20 the ventral processes. Here we performed a phylogenetically controlled comparison of sperm 21 morphology across 44 species of hydromyine rodents to test the hypothesis that the length and 22angle of both the ventral processes and apical hook, as well as the dimensions of the sperm tail, 23 increase with relative testes mass as a proxy for differences in levels of intermale sperm 24 competition. Although both sperm head protrusions exhibited considerable variation in their length 25 and angle across species, only the angles increased significantly in relation to relative testes mass. 26Significant positive relationships were also evident between relative testes mass and lengths of the 27 sperm midpiece and flagellum. These results suggest that in the sperm head of hydromyine rodents, 28 the angle of the ventral processes, as well as that of the apical hook, together with the sperm tail 29 length, are likely to be under sexual selection. The possible functional significance of these findings 30 is discussed.

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Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non‐invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell‐safe with no inherent cell toxicity or light‐induced phototoxicity indicated by negative DNA damage staining. Pre‐measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01‐0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

The high diversity of native Philippine murid rodents includes an old endemic group, the chrotomyines, which are the sister group of the Australasian hydromyines. Herein we detail their interspecific diversity of relative testes mass (RTM) and sperm morphology. We find that in chrotomyines, as in the Australasian hydromyines, testes mass relative to body mass differs by an order of magnitude across the species and ranges from a large RTM in Soricomys and Chrotomys species to a small RTM in Apomys. Sperm morphology is associated with these findings, with individuals in species of Soricomys and Chrotomys producing relatively larger spermatozoa with a prominent apical hook and long tail, whereas, by contrast, the Apomys species have a sperm head that either has a very short or no apical hook and a shorter tail. These findings indicate coevolution of RTM with sperm morphological traits across the species, with the marked interspecific differences in RTM suggesting differences in the intensity of intermale sperm competition and hence breeding system. Thus, we hypothesise that species of Soricomys and Chrotomys that produce more streamlined spermatozoa with longer tails have a polyandrous or promiscuous mating system, whereas the Apomys species, which produce smaller and less streamlined spermatozoa, may exhibit monogamy.

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