Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes; small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of >350 microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes >50 miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation.
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.
The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.
FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1Δ/Δ). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1Δ/Δ mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1Δ/Δ oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1Δ/Δ oocytes, whereas securin and CDC25B, two other APC/CFZR1 substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1Δ/Δ oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/CFZR1 activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.
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.
SUMMARYWithin the mammalian ovary, oocytes remain arrested at G2 for several years. Then a peri-ovulatory hormonal cue triggers meiotic resumption by releasing an inhibitory phosphorylation on the kinase Cdk1. G2 arrest, however, also requires control in the concentrations of the Cdk1-binding partner cyclin B1, a process achieved by anaphase-promoting complex (APC Cdh1 ) activity, which ubiquitylates and so targets cyclin B1 for degradation. Thus, APC Cdh1 activity prevents precocious meiotic entry by promoting cyclin B1 degradation. However, it remains unresolved how cyclin B1 levels are suppressed sufficiently to maintain arrest but not so low that they make oocytes hormonally insensitive. Here, we examined spatial control of this process by determining the intracellular location of the proteins involved and using nuclear-targeted cyclin B1. We found that raising nuclear cyclin B1 concentrations, an event normally observed in the minutes before nuclear envelope breakdown, was a very effective method of inducing the G2/M transition. Oocytes expressed only the a-isoform of Cdh1, which was predominantly nuclear, as were Cdc27 and Psmd11, core components of the APC and the 26S proteasome, respectively. Furthermore, APC Cdh1 activity appeared higher in the nucleus, as nuclear-targeted cyclin B1 was degraded at twice the rate of wild-type cyclin B1. We propose a simple spatial model of G2 arrest in which nuclear APC Cdh1 -proteasomal activity guards against any cyclin B1 accumulation mediated by nuclear import.
(2014) Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice, Cell Cycle, 13:12, 1938Cycle, 13:12, -1947
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