Mitochondrial Dynamics Markers and Related Signaling Molecules Are Important Regulators of Spermatozoa Number and Functionality

Abstract

Here, we study possible mechanisms of (in/sub)fertility related to the acute or repeated psychological stresses (the most common stresses in human society) by following the transcriptional profile of 22 mitochondrial dynamics/function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality. An in vivo study mimicking acute (once for 3 h) and repeated (3 h for 10 consecutive days) psychophysical stress was performed on adult rats. The analysis of hormones, the number/functionality of spermatozoa, and 44 transcriptional markers were performed on individual samples from up to 12 animals per group. Results showed that both types of stress reduced spermatozoa functionality (acute by 4.4-fold, repeated by 3.3-fold) and ATP production (acute by 2.3-fold, repeated by 14.5-fold), while only repeated stress reduces the number of spermatozoa (1.9-fold). Stress significantly disturbed transcription of 34-out-of-44 markers (77%). Mitochondrial dynamics and functionality markers: 18-out-of-22 =>82% (mitochondrial-biogenesis-markers –>6-out-of-8 =>75%; mitochondrial-fusion-markers –>3-out-of-3 =>100%; mitochondrial-fission-markers –>1-out-of-2 =>50%; mitochondrial-autophagy-markers –>3-out-of-3 =>100%; mitochondrial-functionality-markers –>5-out-of-6 =>83%). Markers of signaling pathways regulating both mitochondrial dynamics/functionality and spermatozoa number/functionality important for male (in/sub)fertility –>16-out-of-22 =>73% (cAMP-signaling-markers –>8-out-of-12 =>67%; MAPK-signaling-markers –>8-out-of-10 =>80%). Accordingly, stress-triggered changes of transcriptional profile of mitochondrial dynamics/functionality markers as well as signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality represent adaptive mechanisms.

Spermatozoal Mitochondrial Dynamics Markers and Other Functionality-Related Signaling Molecules Exert Circadian-like Response to Repeated Stress of Whole Organism

“…All experiments were performed in the Laboratory for Reproductive Endocrinology and Signaling and Laboratory for Chronobiology and Aging, Faculty of Sciences at University of Novi Sad (https://wwwold.dbe.pmf.uns.ac.rs/en/nauka-eng/lares, accessed on 4 March 2022). All the methods used in this study were previously reported by our group (for all references, please see [21,22,36]) and followed the relevant guidelines and regulations.…”

“…Animals were bred and raised in the accredited Animal Facility of the Faculty of Sciences, University of Novi Sad, Serbia, in controlled environmental conditions (22 ± 2 • C; 14 h light and 10 h dark cycle, lights on at 7:00 a.m.) with food and water ad libitum. The experimental model of psychophysical stress by immobilization was performed using the method previously described [20][21][22]. In short, stressed (3hIMO) rats were bound in a supine position to a wooden board by fixing the rats' limbs using thread, while the head motion was not limited.…”

“…The level of androgens, in serum, was referred to as T+DHT considering that the antitestosterone serum №250 showed 100% cross-reactivity with DHT (for references, please see [20,22]). Serum androgen levels were measured via radioimmunoassay.…”

“…The isolation of caudal epididymides spermatozoa was carried out following the WHO laboratory manual (https://www.who.int/reproductivehealth/publications/infertility/ 9789241547789/en/, accessed on 4 March 2022) with modifications for rat spermatozoa isolation (for references, please see [21,22]). In short, caudal epididymides were quickly isolated, and surrounding adipose tissue was removed.…”

“…Stressful life has been recognized as one of the main reasons for male infertility [17][18][19], which is a global problem showing an increase in unexplained cases of infertile young males. Different types of stressors and stressful life events have been linked to reduced male reproductive function [18,[20][21][22]. A high number of stressful life events are observed in infertile men, and this was associated with a decline in semen quality during fertility treatment [18].…”