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.

Quiescent sperm survive in cauda epididymis for long periods of time under extreme crowding conditions and with a very limited energy substrate, while after ejaculation, motile sperm live for a much shorter period with an unlimited energy resource and without crowding. Thus, the energy metabolism in relation to the energy requirement of the two may be quite different. A simple physiological technique was evolved to collect viable quiescent sperm from rat cauda epididymis to compare its energy metabolism with motile sperm. Quiescent sperm exhibited 40%-60% higher activities of mitochondrial electron transport chain complexes I-IV and ATP synthase in comparison to motile sperm and accumulated Ca(2+) in the midpiece mitochondria to enhance oxidative phosphorylation (OxPhos). In contrast, motile sperm displayed up to 75% higher activities of key glycolytic enzymes and secreted more than two times the lactate than quiescent sperm. Quiescent sperm phosphorylated AMPK and MAPK-p38, while motile sperm phosphorylated AKT and MAPK/ERK. Glycolytic inhibitor iodoacetamide prevented motility activation of quiescent rat sperm and inhibited conception in rabbits more effectively than OxPhos uncoupler 2,4-dinitrophenol. Apparently, quiescent sperm employ the most energy efficient OxPhos to survive for extended periods of time under extreme conditions of nutrition and crowding. However, on motility initiation, sperm switch predominantly to glycolysis to cater to their high- and quick-energy requirement of much shorter periods. This study also presents a proof of concept for targeting sperm energy metabolism for contraception.

Meiosis is the defining event of spermatogenesis. Spermatocytes undergo meiosis to give rise to round spermatids, which in turn metamorphose to flagellated spermatozoa that mature in the epididymis. To characterize the dynamics of gene expression during these important stages of spermatogenesis, we undertook transcriptome analysis in >90% pure pachytene spermatocytes and round spermatids, and pure mature sperm of rat by massive parallel deep sequencing. The study has identified 10,719 total transcripts expressed in meiotic and postmeiotic cells, out of which 7,641 were present in all the three cell types. Most abundant transcripts were related to gametogenesis in spermatocytes and spermatids, and mitochondrial energy metabolism in sperm. Importantly, 108 transcripts were specific to spermatocytes, including Cpeb2, Dpf3, H2afy, Haus7, Plcb1, Taf9, and Tdrd7 strongly linked with meiosis. Similarly, 323 transcripts unique to round spermatids included Arpc5, Apoa1, Cntrob, Dcaf17, Ift88, and Ly6k that play essential roles in spermiogenesis. Likewise, 178 transcripts unique to sperm included Camta1, Hoxb1, and Prdx6 having assigned roles in fertility and/or embryonic development. Levels of ~16% transcripts declined from spermatocytes to sperm while two (Cd300e and Ddx17) increased. New candidate genes with possible roles in meiosis (91), spermiogenesis (298), and sperm function (171), have been identified. This study has provided new potential targets for contraception and/or treatment of male infertility. (CDRI communication number 9889).

The study was funded by CSIR-Network Project 'PROGRAM' (BSC0101) and partly by the Ministry of Health and Family Welfare, Government of India (GAP0001). The funding agencies did not play any role in this study and none of the authors had any competing interest(s).

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