Embryonic microRNAs are essential for bovine preimplantation embryo development.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

A dynamic basal complex modulates mammalian sperm movement.

Reproductive success depends on efficient sperm movement driven by axonemal dynein-mediated microtubule sliding. Models predict sliding at the base of the tail - the centriole - but such sliding has never been observed. Centrioles are ancient organelles with a conserved architecture; their rigidity is thought to restrict microtubule sliding. Here, we show that, in mammalian sperm, the atypical distal centriole (DC) and its surrounding atypical pericentriolar matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. During asymmetric tail beating, the DC's right side and its surroundings slide ~300 nm rostrally relative to the left side. The deformation throughout the DBC is transmitted to the head-tail junction; thus, the head tilts to the left, generating a kinking motion. These findings suggest that the DBC evolved as a dynamic linker coupling sperm head and tail into a single self-coordinated system.

Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) Finds Patient Age and Sperm Morphology Are Associated With Centriole Quality.

A large proportion of infertility and miscarriage causes are unknown. One potential cause is a defective sperm centriole, a subcellular structure essential for sperm motility and embryonic development. Yet, the extent to which centriolar maladies contribute to male infertility is unknown due to the lack of a convenient way to assess centriole quality. We developed a robust, location-based, ratiometric assay to overcome this roadblock, the Fluorescence-based Ratiometric Assessment of Centrioles (FRAC). We performed a case series study with semen samples from 33 patients, separated using differential gradient centrifugation into higher-grade (pellet) and lower-grade (interface) sperm fractions. Using a reference population of higher-grade sperm from infertile men with morphologically standard sperm, we found that 79% of higher-grade sperm of infertile men with substandard sperm morphology have suboptimal centrioles (P = 0.0005). Moreover, tubulin labeling of the sperm distal centriole correlates negatively with age (P = 0.004, R = -0.66). These findings suggest that FRAC is a sensitive method and that patient age and sperm morphology are associated with centriole quality.

The Role of Sperm Centrioles in Human Reproduction - The Known and the Unknown.

Each human spermatozoon contains two remodeled centrioles that it contributes to the zygote. There, the centrioles reconstitute a centrosome that assembles the sperm aster and participate in pronuclei migration and cleavage. Thus, centriole abnormalities may be a cause of male factor infertility and failure to carry pregnancy to term. However, the precise mechanisms by which sperm centrioles contribute to embryonic development in humans are still unclear, making the search for a link between centriole abnormalities and impaired male fecundity particularly difficult. Most previous investigations into the role of mammalian centrioles during fertilization have been completed in murine models; however, because mouse sperm and zygotes appear to lack centrioles, these studies provide information that is limited in its applicability to humans. Here, we review studies that examine the role of the sperm centrioles in the early embryo, with particular emphasis on humans. Available literature includes case studies and case-control studies, with a few retrospective studies and no prospective studies reported. This literature has provided some insight into the morphological characteristics of sperm centrioles in the zygote and has allowed identification of some centriole abnormalities in rare cases. Many of these studies suggest centriole involvement in early embryogenesis based on phenotypes of the embryo with only indirect evidence for centriole abnormality. Overall, these studies suggest that centriole abnormalities are present in some cases of sperm with asthenoteratozoospermia and unexplained infertility. Yet, most previously published studies have been restricted by the laborious techniques (like electron microscopy) and the limited availability of centriolar markers, resulting in small-scale studies and the lack of solid causational evidence. With recent progress in sperm centriole biology, such as the identification of the unique composition of sperm centrioles and the discovery of the atypical centriole, it is now possible to begin to fill the gaps in sperm centriole epidemiology and to identify the etiology of sperm centriole dysfunction in humans.

Poc1B and Sas-6 Function Together during the Atypical Centriole Formation in Drosophila melanogaster.

Insects and mammals have atypical centrioles in their sperm. However, it is unclear how these atypical centrioles form. Drosophila melanogaster sperm has one typical centriole called the giant centriole (GC) and one atypical centriole called the proximal centriole-like structure (PCL). During early sperm development, centriole duplication factors such as Ana2 and Sas-6 are recruited to the GC base to initiate PCL formation. The centriolar protein, Poc1B, is also recruited at this initiation stage, but its precise role during PCL formation is unclear. Here, we show that Poc1B recruitment was dependent on Sas-6, that Poc1B had effects on cellular and PCL Sas-6, and that Poc1B and Sas-6 were colocalized in the PCL/centriole core. These findings suggest that Sas-6 and Poc1B interact during PCL formation. Co-overexpression of Ana2 and Sas-6 induced the formation of ectopic particles that contained endogenous Poc1 proteins and were composed of PCL-like structures. These structures were disrupted in Poc1 mutant flies, suggesting that Poc1 proteins stabilize the PCL-like structures. Lastly, Poc1B and Sas-6 co-overexpression also induced the formation of PCL-like structures, suggesting that they can function together during the formation of the PCL. Overall, our findings suggest that Poc1B and Sas-6 function together during PCL formation.

It takes two (centrioles) to tango.

Cells that divide during embryo development require precisely two centrioles during interphase and four centrioles during mitosis. This precise number is maintained by allowing each centriole to nucleate only one centriole per cell cycle (i.e. centriole duplication). Yet, how the first cell of the embryo, the zygote, obtains two centrioles has remained a mystery in most mammals and insects. The mystery arose because the female gamete (oocyte) is thought to have no functional centrioles and the male gamete (spermatozoon) is thought to have only one functional centriole, resulting in a zygote with a single centriole. However, recent studies in fruit flies, beetles and mammals, including humans, suggest an alternative explanation: spermatozoa have a typical centriole and an atypical centriole. The sperm typical centriole has a normal structure but distinct protein composition, whereas the sperm atypical centriole is distinct in both. During fertilization, the atypical centriole is released into the zygote, nucleates a new centriole and participates in spindle pole formation. Thus, the spermatozoa's atypical centriole acts as a second centriole in the zygote. Here, we review centriole biology in general and especially in reproduction, we describe the discovery of the spermatozoon atypical centriole, and we provide an updated model for centriole inherence during sexual reproduction. While we focus on humans and other non-rodent mammals, we also provide a broader evolutionary perspective.

Author Correction: A novel atypical sperm centriole is functional during human fertilization.

In the original version of this Article, the affiliation details for Jadranka Loncarek and Vito Mennella were incorrectly given as 'Cell Biology Program, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, ON, M5G 1X8, Canada' and 'Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, 1050 Boyles Street, Frederick, MD, 21702, USA', respectively. This has now been corrected in both the PDF and HTML versions of the Article.

A novel atypical sperm centriole is functional during human fertilization.

The inheritance of the centrosome during human fertilization remains mysterious. Here we show that the sperm centrosome contains, in addition to the known typical barrel-shaped centriole (the proximal centriole, PC), a surrounding matrix (pericentriolar material, PCM), and an atypical centriole (distal centriole, DC) composed of splayed microtubules surrounding previously undescribed rods of centriole luminal proteins. The sperm centrosome is remodeled by both reduction and enrichment of specific proteins and the formation of these rods during spermatogenesis. In vivo and in vitro investigations show that the flagellum-attached, atypical DC is capable of recruiting PCM, forming a daughter centriole, and localizing to the spindle pole during mitosis. Altogether, we show that the DC is compositionally and structurally remodeled into an atypical centriole, which functions as the zygote's second centriole. These findings now provide novel avenues for diagnostics and therapeutic strategies for male infertility, and insights into early embryo developmental defects.

Atypical centrioles during sexual reproduction.

Centrioles are conserved, self-replicating, microtubule-based, 9-fold symmetric subcellular organelles that are essential for proper cell division and function. Most cells have two centrioles and maintaining this number of centrioles is important for animal development and physiology. However, how animals gain their first two centrioles during reproduction is only partially understood. It is well established that in most animals, the centrioles are contributed to the zygote by the sperm. However, in humans and many animals, the sperm centrioles are modified in their structure and protein composition, or they appear to be missing altogether. In these animals, the origin of the first centrioles is not clear. Here, we review various hypotheses on how centrioles are gained during reproduction and describe specialized functions of the zygotic centrioles. In particular, we discuss a new and atypical centriole found in sperm and zygote, called the proximal centriole-like structure (PCL). We also discuss another type of atypical centriole, the "zombie" centriole, which is degenerated but functional. Together, the presence of centrioles, PCL, and zombie centrioles suggests a universal mechanism of centriole inheritance among animals and new causes of infertility. Since the atypical centrioles of sperm and zygote share similar functions with typical centrioles in somatic cells, they can provide unmatched insight into centriole biology.