Janet E. Holt

The MicroRNA Signature of Mouse Spermatozoa Is Substantially Modified During Epididymal Maturation

ABSTRACT 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 Control of Meiotic Maturation in Mammalian Oocytes

Mammalian oocytes spend the majority of their lives in a dormant state, residing in primordial follicles. This arrest, most analogous to the G2 stage of the mitotic cell cycle division, is only broken in the hours preceding ovulation, when a hormonal rise induces meiotic resumption and entry into the first meiotic division. At a molecular level, this event is triggered by CDK1 activity, and here, we examine how CDK1 is suppressed during meiotic arrest and raised for oocyte maturation. We focus on signaling: intercellular signaling between the oocyte and the somatic cells of the follicle, and spatial signaling involving the anaphase-promoting complex (APC) within the oocyte. Meiotic arrest is achieved through APC(FZR1)-mediated cyclin B1 degradation. Once meiotic resumption resumes, CDK1 levels rise, but its activity eventually needs to be suppressed for completion of the first meiotic division. This is achieved by APC(CDC20), whose activity is critically regulated by the spindle assembly checkpoint, and which induces both a loss in CDK1 activity as well as the cohesive ties holding chromosomes together.

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 (APCCdh1) activity, which ubiquitylates and so targets cyclin B1 for degradation. Thus, APCCdh1 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 α-isoform of Cdh1, which was predominantly nuclear, as were Cdc27 and Psmd11, core components of the APC and the 26S proteasome, respectively. Furthermore, APCCdh1 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 APCCdh1-proteasomal activity guards against any cyclin B1 accumulation mediated by nuclear import.

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 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 (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.

Biosynthesis of the Canine Zona Pellucida Requires the Integrated Participation of Both Oocytes and Granulosa Cells

Abstract In the dog, attempts to localize the expression of zona pellucida (ZP) proteins during folliculogenesis have failed to demonstrate conclusively whether any or all of the zona proteins are synthesized in the oocyte or the granulosa cells. Probing of paraformaldehyde-fixed prepubertal canine ovarian tissue sections with a panel of fluorescently conjugated lectins localized the expression of glycoproteins during folliculogenesis. We confirm that six lectins (PSA, s-WGA, ECL, GSL-II, LEL, and STL) consistently labeled the ZP and adjacent granulosa cells of the developing follicle and that canine ZP expresses β-gal(1,4)glcNAc, β-gal(1,3)galNac, α-mannose, and terminal sialic acid residues in a developmentally specific manner. Riboprobes for canine ZPA and ZPC genes were produced and used for in situ hybridization studies of mRNA expression in canine folliculogenesis. In addition, we isolated a partial cDNA transcript from total ovarian RNA for the canine ZPB gene having a high degree of sequence identity with the felid and porcine ZPB homologues. Subsequently, the ZPA gene transcripts were localized to the cytoplasm of oocytes in primordial, primary, and early secondary follicles. We then localized expression of ZPB and ZPC gene transcripts to the granulosa cells of growing follicles, but not in squamous granulosa cells of primordial follicles or oocytes. These observations indicate that in the juvenile canine ovary, the oocyte is responsible for synthesis of the ZPA protein and directing synthesis of the ZPB and ZPC proteins by the granulosa cells and that ZP gene transcription occurs in a sequential manner during folliculogenesis.

Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice

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.

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.

APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

The APC activator FZR1 has a role in controlling the timing of meiosis I spindle assembly. Oocytes lacking FZR1 undergo accelerated meiosis I, associated with earlier spindle assembly checkpoint satisfaction and APCCDC20 activity, resulting in high rates of aneuploidy.

Importin Alpha2-Interacting Proteins with Nuclear Roles During Mammalian Spermatogenesis

ABSTRACT 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.