The thermo-sensitive gene expression signatures of spermatogenesis

Abstract

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.

Endoplasmic Reticulum Stress (ER Stress) and Unfolded Protein Response (UPR) Occur in a Rat Varicocele Testis Model

“…Testicular sections fixed by Bouin were evaluated after staining with haematoxylin/eosin (HE) and parameters such as mean percentage of tubular diameter, epithelial height, meiotic index, percentage of spermatogenesis, and Johnsen's score [ 29 ] were evaluated using an Olympus CX31 microscope (40x magnification). The TUNEL stain was used as previously indicated for the evaluation of apoptosis [ 30 ].…”

The dynamics of gene expression during and post meiosis sets the sperm agenda

“…The viability of the purified cells in the two fractions was >95% by trypan blue exclusion test. The details of purified cells have been published earlier (Yadav et al, ).…”

“…Likewise, spink2 deficient mouse have increased serine protease activity, disrupted spermatogenesis, and germ cell apoptosis (B. Lee et al, ), while the spink2 knockout homozygous male mice are azoospermic (Kherraf et al, ). The Slc16a7 (monocarboxylate transporter‐2) that transports lactate from Sertoli to germ cells was previously reported predominantly in spermatocytes and spermatids (Yadav et al, ) and genetic variations in MCT‐2 have clinical relevance in male infertility (J. Lee, Lee, & Lee, ). Similarly, the Usp9y is located in the AZFa region of Y chromosome whose deletion causes azoospermia or Sertoli cell only syndrome, and partial deletion of AZFa (USP9Y gene) can result in oligozoospermia (Song, Chiba, Ramasamy, & Lamb, ).…”

“…The purity of isolated cells (~90%) was checked visually under a microscope and through DNA quantitation using flow cytometry. The details of cell isolation, purification and purity check have been published earlier (Yadav et al, ). Alternatively, live cells were also stained at room temperature with acridine orange (100 nM) in BME for 5 min at room temperature and visualized for nuclear morphology and cell purity under a fluorescence microscope (Nikon 80i) using B2A filter and 40X objective.…”

“…The Mgat1 gene encodes the enzyme N-acetylglucosaminyltransferase that regulates ERK signaling (Biswas, Batista, Sundaram, & Stanley, 2018), which is indispensable for spermatogenesis and Mgat1 deletion in spermatogonia did not produce any sperm (Batista, Lu, Williams, & Stanley, 2012 azoospermic (Kherraf et al, 2017). The Slc16a7 (monocarboxylate transporter-2) that transports lactate from Sertoli to germ cells was previously reported predominantly in spermatocytes and spermatids (Yadav et al, 2018) and genetic variations in MCT-2 have clinical relevance in male infertility (J. Lee, Lee, & Lee, 2014).…”