Dynamin 2-dependent endocytosis is essential for mouse oocyte development and fertility.

During folliculogenesis, oocytes are dependent on metabolic and molecular support from surrounding somatic cells. Here, we examined the role of the dynamin (DNM) family of mechanoenzymes in mediating endocytotic uptake into growing follicular oocytes. We found DNM1 and DNM2 to be highly expressed in growing follicular oocytes as well as in mature germinal vesicle (GV) and metaphase II (MII) stage oocytes. Moreover, oocyte-specific conditional knockout (cKO) of DNM2 (DNM2Δ) led to complete sterility, with follicles arresting at the preantral stage of development. In addition, DNM2Δ ovaries were characterized by disrupted follicular growth as well as oocyte and follicle apoptosis. Further, the loss of DNM activity, either through DNM2 cKO or through pharmacological inhibition (Dyngo 6a) led to the impairment of endocytotic pathways in preantral oocytes as well as in mature GV and MII oocytes, respectively. Loss of DNM activity resulted in the redistribution of endosomes and the misslocalization of clathrin and actin, suggesting dysfunctional endocytosis. Notably, there was no observable effect on the fertility of DNM1Δ females. Our study has provided new insight into the complex and dynamic nature of oocyte growth during folliculogenesis, suggesting a role for DNM2 in mediating the endocytotic events that are essential for oocyte development.

The APC/C activator FZR1 is essential for meiotic prophase I in mice.

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

Maternal Smoke Exposure Impairs the Long-Term Fertility of Female Offspring in a Murine Model.

The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood; however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.

The microRNA signature of mouse spermatozoa is substantially modified during epididymal 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.

Changing expression and subcellular distribution of karyopherins during murine oogenesis.

Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined (Kpna1, Kpna2, Kpna3, Kpna4, Kpna6, Kpna7, Kpnb1, Ipo5 and Xpo1), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1, Kpna2, Kpna4, Kpna6 and Ipo5 and down-regulation of Kpnb1, Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs--KPNA1, KPNA2 and IPO5--displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development.

Furin processing dictates ectodomain shedding of human FAT1 cadherin.

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.

The APC activator fizzy-related-1 (FZR1) is needed for preimplantation mouse embryo development.

In early embryos of a number of species the anaphase-promoting complex (APC), an important cell cycle regulator, requires only CDC20 for cell division. In contrast, fizzy-related-1 (FZR1), a non-essential protein in many cell types, is thought to play a role in APC activation at later cell cycles, and especially in endoreduplication. In keeping with this, Fzr1 knockout mouse embryos show normal preimplantation development but die due to a lack of endoreduplication needed for placentation. However, interpretation of the role of FZR1 during this period is hindered by the presence of maternal stores. In this study, therefore, we used an oocyte-specific knockout to examine FZR1 function in early mouse embryo development. Maternal FZR1 was not crucial for completion of meiosis, and furthermore viable pups were born to Fzr1 knockout females mated with normal males. However, in early embryos the absence of both maternal and paternal FZR1 led to a dramatic loss in genome integrity, such that the majority of embryos arrested having undergone only a single mitotic division and contained many γ-H2AX foci, consistent with fragmented DNA. A prominent feature of such embryos was the establishment of two independent spindles following pronuclear fusion and thus a failure of the chromosomes to mix (syngamy). These generated binucleate 2-cell embryos. In the 10% of embryos that progressed to the 4-cell stage, division was so slow that compaction occurred prematurely. No embryo development to the blastocyst stage was ever observed. We conclude that Fzr1 is a surprisingly essential gene involved in the establishment of a single spindle from the two pronuclei in 1-cell embryos as well as being involved in the maintenance of genomic integrity during the mitotic divisions of early mammalian embryos.

The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes.

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 (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/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 Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) 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 Fzr1(Δ/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/C(FZR1) 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.

Through the smoke: use of in vivo and in vitro cigarette smoking models to elucidate its effect on female fertility.

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.

Spatial regulation of APCCdh1-induced cyclin B1 degradation maintains G2 arrest in mouse oocytes.

Within 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 alpha-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.

Reduced chromosome cohesion measured by interkinetochore distance is associated with aneuploidy even in oocytes from young mice.

It is becoming clear that reduced chromosome cohesion is an important factor in the rise of maternal age-related aneuploidy. This reduction in cohesion has been observed both in human and mouse oocytes, and it can be measured directly by an increase with respect to maternal age in interkinetochore (iKT) distance between a sister chromatid pair. We have observed variations in iKT distance even in oocytes from young mice and wondered if such differences may predispose those oocytes displaying the greatest iKT distances to be becoming aneuploid. Therefore, we used two methods, one pharmacological (Aurora kinase inhibitor) and one genetic (Fzr1 knockout), to raise aneuploidy rates in oocytes from young mice (age, 1-3 mo) and to examine if those oocytes that were aneuploid had greater iKT distances. We observed that for both Aurora kinase inhibition and Fzr1 knockout, iKT distances were significantly greater in those oocytes that became aneuploid compared to those that remained euploid. Based on these results, we propose that individual oocytes undergo loss in chromosomal cohesion at different rates and that the greater this loss, the greater the risk for becoming aneuploid.

Importin alpha2-interacting proteins with nuclear roles during mammalian spermatogenesis.

Spermatogenesis, the process of generating haploid sperm capable of fertilizing the female gamete, requires the timely transport into the nucleus of transcription and chromatin-remodeling factors, mediated by members of the importin (IMP) superfamily. Previous IMP expression profiling implies a role for IMPalpha2 in testicular germ cells late in spermatogenesis. To identify interacting proteins of IMPalpha2 that are potential drivers of germ cell development, we performed yeast two-hybrid screening of an adult mouse testis library. IMPalpha2 interactions were verified by coimmunoprecipitation approaches, whereas immunohistochemical staining of testis sections confirmed their coexpression with IMPalpha2 in specific testicular cell types. Key interactors identified were a novel isoform of a cysteine and histidine rich protein (Chrp), a protein inhibitor of activated STAT (PIAS) family member involved in transcriptional regulation and sumoylation, Androgen receptor interacting protein 3 (Arip3), and Homologous protein 2 (Hop2), known to be involved in homologous chromosome pairing and recombination, all of which are highly expressed in the testis and show mRNA expression profiles similar to that of IMPalpha2 throughout testicular development. This is the first study to identify binding partners of IMPalpha2 in the developmental context of germ line development, and we propose that the regulated expression and timely IMPalpha2-mediated nuclear transport of these proteins may coordinate events during spermatogenesis, with IMPalpha2-mediated nuclear localization representing a potentially critical developmental switch in the testis.

Control of homologous chromosome division in the mammalian oocyte.

Homologous chromosomes are segregated during the first meiotic division (meiosis I). Unfortunately, human oocytes are particularly susceptible to mis-segregation errors, so generating aneuploid, often non-viable, embryos. Here we review the cell biology of meiosis I and how homolog disjunction is regulated for mammalian oocytes. We focus on the activity of the anaphase-promoting complex/cyclosome (APC/C), which is responsible for timely degradation of the cohesin component, REC8 and the cyclin B regulatory subunit of maturation-promoting factor, both essential steps for meiosis I completion. In particular, we examine the role played by the spindle assembly checkpoint in controlling the APC/C activity, and in so doing ensuring accurate disjunction of homologs.

The small non-coding RNA profile of mouse oocytes is modified during aging.

Oocytes are reliant on messenger RNA (mRNA) stores to support their survival and integrity during a protracted period of transcriptional dormancy as they await ovulation. Oocytes are, however, known to experience an age-associated alteration in mRNA transcript abundance, a phenomenon that contributes to reduced developmental potential. Here we have investigated whether the expression profile of small non-protein-coding RNAs (sRNAs) is similarly altered in aged mouse oocytes. The application of high throughput sequencing revealed substantial changes to the global sRNA profile of germinal vesicle stage oocytes from young (4-6 weeks) and aged mice (14-16 months). Among these, 160 endogenous small-interfering RNAs (endo-siRNAs) and 10 microRNAs (miRNAs) were determined to differentially accumulate within young and aged oocytes. Further, we revealed decreased expression of two members of the kinesin protein family, Kifc1 and Kifc5b, in aged oocytes; family members selectively targeted for expression regulation by endo-siRNAs of elevated abundance. The implications of reduced Kifc1 and Kifc5b expression were explored using complementary siRNA-mediated knockdown and pharmacological inhibition strategies, both of which led to increased rates of aneuploidy in otherwise healthy young oocytes. Collectively, our data raise the prospect that altered sRNA abundance, specifically endo-siRNA abundance, could influence the quality of the aged oocyte.

Motoring through: the role of kinesin superfamily proteins in female meiosis.

BACKGROUND: The kinesin motor protein family consists of 14 distinct subclasses and 45 kinesin proteins in humans. A large number of these proteins, or their orthologues, have been shown to possess essential function(s) in both the mitotic and the meiotic cell cycle. Kinesins have important roles in chromosome separation, microtubule dynamics, spindle formation, cytokinesis and cell cycle progression. This article contains a review of the literature with respect to the role of kinesin motor proteins in female meiosis in model species. Throughout, we discuss the function of each class of kinesin proteins during oocyte meiosis, and where such data are not available their role in mitosis is considered. Finally, the review highlights the potential clinical importance of this family of proteins for human oocyte quality. OBJECTIVE AND RATIONALE: To examine the role of kinesin motor proteins in oocyte meiosis. SEARCH METHODS: A search was performed on the Pubmed database for journal articles published between January 1970 and February 2017. Search terms included 'oocyte kinesin' and 'meiosis kinesin' in addition to individual kinesin names with the terms oocyte or meiosis. OUTCOMES: Within human cells 45 kinesin motor proteins have been discovered, with the role of only 13 of these proteins, or their orthologues, investigated in female meiosis. Furthermore, of these kinesins only half have been examined in mammalian oocytes, despite alterations occurring in gene transcripts or protein expression with maternal ageing, cryopreservation or behavioral conditions, such as binge drinking, for many of them. WIDER IMPLICATIONS: Kinesin motor proteins have distinct and important roles throughout oocyte meiosis in many non-mammalian model species. However, the functions these proteins have in mammalian meiosis, particularly in humans, are less clear owing to lack of research. This review brings to light the need for more experimental investigation of kinesin motor proteins, particularly those associated with maternal ageing, cryopreservation or exposure to environmental toxicants.

Kif4 Is Essential for Mouse Oocyte Meiosis.

Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

Germ cell specific overactivation of WNT/ßcatenin signalling has no effect on folliculogenesis but causes fertility defects due to abnormal foetal development.

All the major components of the WNT signalling pathway are expressed in female germ cells and embryos. However, their functional relevance in oocyte biology is currently unclear. We examined ovaries collected from TCFGFP mice, a well-known Wnt reporter mouse model, and found dynamic changes in the Wnt/ßcatenin signalling activity during different stages of oocyte development and maturation. To understand the functional importance of Wnt signalling in oocytes, we developed a mouse model with the germ cell-specific constitutive activation of ßcatenin using cre recombinase driven by the DEAD (Asp-Glu-Ala-Asp) box protein 4 (Ddx4) gene promoter. Histopathological and functional analysis of ovaries from these mutant mice (Ctnnb1(ex3)cko) showed no defects in ovarian functions, oocytes, ovulation and early embryonic development. However, breeding of the Ctnnb1(ex3)cko female mice with males of known fertility never resulted in birth of mutant pups. Examination of uteri from time pregnant mutant females revealed defects in ectoderm differentiation leading to abnormal foetal development and premature death. Collectively, our work has established the role of active WNT/ßcatenin signalling in oocyte biology and foetal development, and provides novel insights into the possible mechanisms of complications in human pregnancy such as repeated spontaneous abortion, sudden intrauterine unexpected foetal death syndrome and stillbirth.

Dynamin 2 is essential for mammalian spermatogenesis.

The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome.

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.