Dr Janet Bristow

The mammalian epididymis is a highly specialized region of the male reproductive tract that is lined with a continuous layer of epithelial cells that display a remarkable level of regionalized secretory and absorptive activity. The luminal environment created by this combined secretory and absorptive activity is directly responsible for promoting the functional maturation of spermatozoa and their maintenance in a quiescent and viable state prior to ejaculation. This study was designed to identify the complement of microRNAs (miRNAs) that are expressed within the mouse epididymal epithelial cells and the maturing populations of spermatozoa. Through the use of Next Generation Sequencing technology we have demonstrated that both epididymal epithelial cells and spermatozoa harbour a complex repertoire of miRNAs that have substantially different expression profiles along the length of the tract. These data, deposited in the Gene Expression Omnibus (GEO) with the accession numbers GSE70197 and GSE70198, afford valuable insight into the post-transcriptional control of gene expression within the epididymis and provide the first evidence for the dynamic transformation of the miRNA content of maturing sperm cells. Ultimately such information promises to inform our understanding of the aetiology of male infertility. Herein we provide a detailed description of the methodology used to generate these important data.

The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract.

In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.

Fat1 is a single pass transmembrane protein and the largest member of the cadherin superfamily. Mouse knockout models and in vitro studies have suggested that Fat1 influences cell polarity and motility. Fat1 is also an upstream regulator of the Hippo pathway, at least in lower vertebrates, and hence may play a role in growth control. In previous work we have established that FAT1 cadherin is initially cleaved by proprotein convertases to form a noncovalently linked heterodimer prior to expression on the cell surface. Such processing was not a requirement for cell surface expression, since melanoma cells expressed both unprocessed FAT1 and the heterodimer on the cell surface. Here we further establish that the site 1 (S1) cleavage step to promote FAT1 heterodimerisation is catalysed by furin and we identify the cleavage site utilised. For a number of other transmembrane receptors that undergo heterodimerisation the S1 processing step is thought to occur constitutively but the functional significance of heterodimerisation has been controversial. It has also been generally unclear as to the significance of receptor heterodimerisation with respect to subsequent post-translational proteolysis that often occurs in transmembrane proteins. Exploiting the partial deficiency of FAT1 processing in melanoma cells together with furin-deficient LoVo cells, we manipulated furin expression to demonstrate that only the heterodimer form of FAT1 is subject to cleavage and subsequent release of the extracellular domain. This work establishes S1-processing as a clear functional prerequisite for ectodomain shedding of FAT1 with general implications for the shedding of other transmembrane receptors. © 2014.

A finite number of oocytes are established within the mammalian ovary prior to birth to form a precious ovarian reserve. Damage to this limited pool of gametes by environmental factors such as cigarette smoke and its constituents therefore represents a significant risk to a woman's reproductive capacity. Although evidence from human studies to date implicates a detrimental effect of cigarette smoking on female fertility, these retrospective studies are limited and present conflicting results. In an effort to more clearly understand the effect of cigarette smoke, and its chemical constituents, on female fertility, a variety of in vivo and in vitro animal models have been developed. This article represents a systematic review of the literature regarding four of experimental model types: 1) direct exposure of ovarian cells and follicles to smoking constituents' in vitro, 2) direct exposure of whole ovarian tissue with smoking constituents in vitro, 3) whole body exposure of animals to smoking constituents and 4) whole body exposure of animals to cigarette smoke. We summarise key findings and highlight the strengths and weaknesses of each model system, and link these to the molecular mechanisms identified in smoke-induced fertility changes.

Currently, maternal aging in women, based on mouse models, is thought to raise oocyte aneuploidy rates, because chromosome cohesion deteriorates during prophase arrest, and Sgo2, a protector of centromeric cohesion, is lost. Here we show that the most common mouse strain, C57Bl6/J, is resistant to maternal aging, showing little increase in aneuploidy or Sgo2 loss. Instead it demonstrates significant kinetochore-associated loss in the spindle assembly checkpoint protein Mad2 and phosphorylated Aurora C, which is involved in microtubule-kinetochore error correction. Their loss affects the fidelity of bivalent segregation but only when spindle organization is impaired during oocyte maturation. These findings have an impact clinically regarding the handling of human oocytes ex vivo during assisted reproductive techniques and suggest there is a genetic basis to aneuploidy susceptibility. © 2014 Landes Bioscience.

The first meiotic division of mammalian oocytes physiologically occurs in the ovary in the hours preceding ovulation. Fortunately, oocytes removed from their follicular environment will readily undergo this process in culture. Their large size, optical transparency, and efficiency in translating exogenous cRNA make mouse oocytes very amenable to study this process in detail using fluorescence imaging-based techniques. Here we describe the process of microinjecting cRNA to proteins of interest that have been coupled to a fluorescent protein using cyclin B1 as an example. © 2013 Springer Science+Business Media, LLC.