Identification of differentially expressed mRNAs and miRNAs in spermatozoa of bulls of varying fertility.

Bulls used in artificial insemination, with apparently normal semen quality, can vary significantly in their field fertility. This study aimed to characterize the transcriptome of spermatozoa from high (HF) and low (LF) fertility bulls at the mRNA and miRNA level in order to identify potential novel markers of fertility. Holstein-Friesian bulls were assigned to either the HF or LF group (n = 10 per group) based on an adjusted national fertility index from a minimum of 500 inseminations. Total RNA was extracted from a pool of frozen-thawed spermatozoa from three different ejaculates per bull, following which mRNA-seq and miRNA-seq were performed. Six mRNAs and 13 miRNAs were found differentially expressed (P < 0.05, FC > 1.5) between HF and LF bulls. Of particular interest, the gene pathways targeted by the 13 differentially expressed miRNAs were related to embryonic development and gene expression regulation. Previous studies reported that disruptions to protamine 1 mRNA (PRM1) had deleterious consequences for sperm chromatin structure and fertilizing ability. Notably, PRM1 exhibited a higher expression in spermatozoa from LF than HF bulls. In contrast, Western Blot analysis revealed a decrease in PRM1 protein abundance for spermatozoa from LF bulls; this was not associated with increased protamine deficiency (measured by the degree of chromatin compaction) or DNA fragmentation, as assessed by flow cytometry analyses. However, protamine deficiency was positively and moderately correlated with the percentage of spermatozoa with DNA fragmentation, irrespective of fertility group. This study has identified potential biomarkers that could be used for improving semen quality assessments of bull fertility.

Imaging and Quantitation of Assembly Dynamics of the Centromeric Histone H3 Variant CENP-A in Drosophila melanogaster Spermatocytes by Immunofluorescence and Fluorescence In-Situ Hybridization (Immuno-FISH).

This chapter describes a method used to assay the cell cycle dynamics of the centromeric histone H3 variant CENP-A in meiosis using Drosophila males as the experimental system. Specifically, we describe a method that combines Immunofluorescence (IF) and Fluorescence in-situ Hybridization (FISH) protocols, performed on fixed Drosophila testes. An advantage of this protocol is the ability to localize individual centromeres on the four Drosophila homologous chromosomes that form distinct nuclear territories in spermatocytes. We also describe a method to quantify CENP-A focal intensities using Image J software.

ATP synthase F1 subunits recruited to centromeres by CENP-A are required for male meiosis.

The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. Here we report an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, we report that knockdown of CENP-A, as well as subunits ATPsyn-α, -ßlike (a testis-specific paralogue of ATPsyn-ß) and -γ disrupts sister centromere cohesion in meiotic prophase I. We find that this disruption is likely independent of reduced ATP levels. We identify that ATPsyn-α and -ßlike localise to meiotic centromeres and that this localisation is dependent on the presence of CENP-A. We show that ATPsyn-α directly interacts with the N-terminus of CENP-A in vitro and that truncation of its N terminus perturbs sister centromere cohesion in prophase I. We propose that the CENP-A N-terminus recruits ATPsyn-α and -ßlike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.

Centromere Dynamics in Male and Female Germ Cells.

In sexually reproducing organisms the germ line is the cellular lineage that gives rise to gametes. All germ cells originate from germline stem cells that divide asymmetrically to generate gonial pre-cursors, which are amplified in number by mitotic divisions, undergo meiosis and eventually differentiate into mature gametes (haploid eggs and sperm). Information transmitted with gametes is inherited by offspring, and potentially by subsequent generations, instructing in organismal development and beyond. Meiosis comprises one round of DNA replication, followed by two rounds of chromosome segregation; homologous chromosomes segregate in the first division (meiosis I) and sister chromatids segregate in the second division (meiosis II). Important mechanistic features of meiosis occur in substages of prophase I and are critical for genetic recombination, including pairing and synapsis of homologous chromosomes (at leptotene and zygotene), crossing-over (at pachytene), and the appearance of chiasmata (at diplotene/diakinesis). Another unique feature of meiosis is the altered centromere/kinetochore geometry at metaphase I, such that sister kinetochores face the same spindle pole (mono-orientation) and stay together at anaphase I. This chapter reviews centromere dynamics in germ cells, focusing on centromere function and assembly in meiotic cell cycles, as well as centromere inheritance in zygotes. Centromeres are functionally defined by the presence of the histone H3 variant CENP-A, the epigenetic determinant of centromere identity. In most eukaryotes, it is well established that CENP-A function is essential for chromosome segregation in mitosis. CENP-A function in meiosis is less well understood and emerging insights into the differential regulation of meiotic and mitotic CENP-A are discussed.

Nucleolar activity and CENP-C regulate CENP-A and CAL1 availability for centromere assembly in meiosis.

The centromere-specific histone CENP-A is the key epigenetic determinant of centromere identity. Whereas most histones are removed from mature sperm, CENP-A is retained to mark paternal centromeres. In Drosophila males we show that the centromere assembly factors CAL1 and CENP-C are required for meiotic chromosome segregation, CENP-A assembly and maintenance on sperm, as well as fertility. In meiosis, CENP-A accumulates with CAL1 in nucleoli. Furthermore, we show that CENP-C normally limits the release of CAL1 and CENP-A from nucleoli for proper centromere assembly in meiotic prophase I. Finally, we show that RNA polymerase I transcription is required for efficient CENP-A assembly in meiosis, as well as centromere tethering to nucleoli.