BackgroundSpermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment.MethodsTestes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs.ResultsHeat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types.ConclusionThe study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.

Objectives To identify the sequence of inflammation‐driven signaling cascades and other molecular events that might cause tumor‐like transformation of prostatic cells. Methods Cytokine array analysis, Reactome and STRING analysis, immunoblotting, and immunocytochemistry were used to investigate the molecular mechanisms governing inflammation‐driven adverse changes in human prostatic cells caused by the sexually transmitted infection, Trichomonas vaginalis, resulting in prostatitis, benign prostatic hyperplasia and prostate cancer. Results Array analysis showed upregulation of 23 cytokines within 24 h of infection of human prostatic epithelial RWPE‐1 cells with the parasite, in vitro. Reactome and STRING analysis of array data identified interleukin‐6, interleukin‐8, nuclear factor kappa B, signal transducer and activator of transcription 3 and cyclooxygenase 2 as chief instigators of prostatic anomaly, which were found to be significantly upregulated by immunofluorescence and western blotting analyses. STRING further connected these instigators with macrophage migration inhibitory factor, PIM‐1 and prostate‐specific antigen; which was confirmed by their marked stimulation in infected prostatic cells by immunoblotting and immunocytochemistry. Upregulated proliferation markers, such as Ki67, proliferating cell nuclear antigen and B‐cell lymphoma 2, suggested tumor‐like signaling in infected RWPE‐1 cells, which was further supported by downregulation of E‐cadherin, upregulation of vimentin and activation of focal adhesion kinase. Prostate tumor DU145 cells were more sensitive to parasite invasion, and showed rapid upregulation with nuclear translocation of sensitive parameters, such as nuclear factor kappa B, signal transducer and activator of transcription 3, and macrophage migration inhibitory factor. The migration of DU145 cells augmented when incubated in spent media from parasite‐infected RWPE‐1 cells. Conclusion The initiation of inflammation driven tumor‐like cell signaling in parasite‐infected human prostatic epithelial cells is apparent, with the prostate tumor (DU145) cells being more sensitive to T. vaginalis than normal (RWPE‐1) prostatic cells.

Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell-survival during storage in cauda epididymis are not known. Intracellular signalling via phosphorylation cascades are quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan® intracellular signalling array provided the preliminary picture of cell-signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signalling-proteins involved in motility activation was constructed by STRING-software, which identified MAPK-p38, AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western-blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signalling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice-versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and ROS levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signalling. Aberrant cell signalling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.