Ejaculated bovine spermatozoa retain a pool of RNAs that may have a function in early embryogenesis and be used as predictors of male fertility. The bovine spermatozoal transcript profile remains incomplete because previous studies have relied on hybridization-based techniques, which evaluate a limited pool of transcripts and cannot identify full-length transcripts. The goal of this study was to sequence the complete cryopreserved bovine spermatozoal transcript profile using Illumina RNA-Sequencing (RNA-Seq). Spermatozoal RNA was pooled from nine bulls with conception rate scores ranging from -2.9 to 3.5 and confirmed to exclude genomic DNA and somatic cell mRNA. After selective amplification of poly(A)(+) RNA and high-throughput sequencing, 6166 transcripts were identified via alignment to the bovine genome (UMD 3.1/bosTau6). RNA-Seq transcript levels (n = 9) were highly correlated with quantitative PCR copy number (r(2) = 0.9747). The bovine spermatozoal transcript profile is a heterogeneous population of degraded and full-length predominantly nuclear-encoded mRNAs. Highly abundant spermatozoal transcripts included PRM1, HMGB4, and mitochondrial-encoded transcripts. Full-length transcripts comprised 66% of the top 368 transcripts (fragments per kilobase of exon per million fragments mapped [FPKM] > 100) and amplification of the full-length transcript or 5' and 3' ends was confirmed for selected transcripts. In addition to the identification of transcripts not previously reported in spermatozoa, several known spermatozoal transcripts from various species were also found. Gene ontology analysis of the FPKM > 100 spermatozoal transcripts revealed that translation was the most predominant biological process represented. This is the first report of the spermatozoal transcript profile in any species using high-throughput sequencing, supporting the presence of mRNA in spermatozoa for further functional and fertility studies.
Improved methods for culturing spermatogenic cells will facilitate the study of spermatogenesis, treatment of male factor infertility, and genetic modification of the male germ line. The objective of this study was to develop a procedure for achieving male germ cell progression through meiosis in vitro. Testes from 3-day-old bulls were decapsulated and seminiferous tubules were dissociated enzymatically to recover Sertoli and germ cells. Dissociated cells were reaggregated by phytohemagglutinin and encapsulated by calcium alginate, then cultured for up to 14 wk in modified Dulbecco modified Eagle medium/F12 (32 degrees C, 5% CO(2) in air). At 2, 5, and 10 wk, cultured cells were examined and evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis for protamine-2 (PRM-2) and transition protein-1 (TP-1) mRNA, expressed specifically in round spermatids. Ploidy was characterized by flow cytometric analysis of DNA content of cultured cells. Only Sertoli cells and gonocytes were observed in seminiferous tubules of 3-day-old testes. By 10 wk of culture, small spherical cells (7-10 microm) were apparent at the margin of cell associations in culture. Following RT-PCR and Northern blot analysis, specific bands corresponding to PRM-2 and TP-1 were detected only in adult testis RNA or after 10 wk of culture. Based on flow cytometry, a haploid population of cells appeared in vitro that was not in 3-day-old bull testis. The novel culture system developed in this study is the first to promote differentiation of gonocytes to presumptive spermatids in vitro based on the expression of spermatid-specific genes.
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Semen from four Holstein bulls was evaluated to compare effects of four extender treatments on postthaw semen quality. Extender fractions A and B, either heated whole milk or 20% egg yolk-citrate, were combined to yield the extender treatments 1) milk and milk, 2) milk and egg yolk-citrate, 3) egg yolk-citrate and milk, and 4) egg yolk-citrate and egg yolk-citrate. Semen was evaluated at thawing and after 30, 60, 120, and 180 min of incubation at 38.5 degrees C. Flow cytometry showed that acridine orange-stained sperm were most susceptible to in situ DNA denaturation when fraction A was milk. For sperm stained with rhodamine 123, flow cytometry showed that the proportion with intact mitochondrial membrane potential was lowest of all treatments at thawing but greatest at 180-min incubation with milk and milk extender. Flow cytometry of propidium iodine-stained sperm showed greatest proportion of cell membrane intact sperm when fraction A was egg yolk-citrate. Light microscopy showed the lowest proportion of cell membrane intact sperm with milk and milk extender after eosin-aniline blue vital staining. Postthaw motility scores tended to be reduced when both extender fractions were egg yolk-citrate. Results demonstrate differential extender effects on postthaw semen quality and indicate that altering extender composition or sequence of adding extender components may improve postthaw quality of cryopreserved sperm.
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