Spermatozoa Develop Molecular Machinery to Recover From Acute Stress

Abstract

This study was designed to search for the possible mechanism(s) of male (in/sub)fertility by following the molecular response of spermatozoa on acute psychological stress (the most common stress in human society) and on a 20-h time-dependent recovery period. To mimic in vivo acute stress, the rats were exposed to immobilization once every 3 h. The recovery periods were as follows: 0 (immediately after stress and 3 h after the light is on-ZT3), 8 (ZT11), 14 (ZT17), and 20 (ZT23) h after stress. Results showed that acute stress provoked effects evident 20 h after the end of the stress period. Numbers of spermatozoa declined at ZT17 and ZT23, while functionality decreased at ZT3 and ZT11, but recovered at ZT17 and ZT23. Transcriptional profiles of 91% (20/22) of tracked mitochondrial dynamics and functionality markers and 91% (20/22) of signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality were disturbed after acute stress and during the recovery period. Most of the changes presented as increased transcription or protein expression at ZT23. The results of the principal component analysis (PCA) showed the clear separation of acute stress recovery effects during active/dark and inactive/light phases. The physiological relevance of these results is the recovered positive-acrosome-reaction, suggesting that molecular events are an adaptive mechanism, regulated by acute stress response signaling. The results of the PCA confirmed the separation of the effects of acute stress recovery on gene expression related to mitochondrial dynamics, cAMP, and MAPK signaling. The transcriptional patterns were different during the active and inactive phases. Most of the transcripts were highly expressed during the active phase, which is expected given that stress occurred at the beginning of the inactive phase. To the best of our knowledge, our results provide a completely new view and the first presentation of the markers of mitochondrial dynamics network in spermatozoa and their correlation with signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality during recovery from acute stress. Moreover, the interactions between the proteins important for spermatozoa homeostasis and functionality (MFN2 and PRKA catalytic subunit, MFN2 and p38MAPK) are shown for the first time. Since the existing literature suggests the importance of semen quality and male fertility not only as the fundamental marker of reproductive health but also as the fundamental biomarkers of overall health and harbingers for the development of comorbidity and mortality, we anticipate our result to be a starting point for more investigations considering the mitochondrial dynamics markers or their transcriptional profiles as possible predictors of (in/sub)fertility.

Keywords: MAPK signaling markers; acute psychological stress; cAMP signaling markers; mitochondrial dynamics and functionality markers; spermatozoa number and functionality; stress recovery.

Figure 1

Experimental design of immobilization stress with recovery period used to assess spermatozoa number and functionality (% acrosome reaction) as well as mitochondrial dynamics markers and related signaling molecule expression profiles of transcripts and proteins.

Figure 2

The acute psychophysical stress by immobilization (IMO) decreases functionality and number of spermatozoa in different time points after the IMO stress. Number of spermatozoa (A) isolated from caudal epididymides of unstressed rats (control) and rats subjected to acute immobilization stress once for 3 h (1x3hIMO) with recovery periods of 0, 8, 14 and 20 h. (B) The functionality of spermatozoa (% of acrosome reacted spermatozoa) isolated from control and acutely (1x3hIMO) stressed rats. Capacitated spermatozoa were stimulated with progesterone (PROG 15 µM) in parallel with spermatozoa not treated with progesterone (PROG 0 µM). Blue staining in the acrosome region of the head indicated intact acrosome, whereas spermatozoa without blue staining in the acrosome region were considered to be acrosome reacted. Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, # vs. the control group of ZT3 time point.

Figure 3

Transcription of mitochondrial biogenesis markers is significantly changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA and proteins from spermatozoa of undisturbed and stressed rats were used for the analysis of the transcriptional profile and protein expression profile of markers of mitochondrial biogenesis. The representative blots are shown as panels. Data from scanning densitometry were normalized on GAPDH (internal control). Values are shown as bars above the photos of blots. Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, ^# vs. the control group of ZT3 time point.

Figure 4

Transcription of mitochondrial fusion and architecture markers, as well as interactions of mitofusin 2 protein and PRKAc and p38 MAPK proteins are significantly changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of mitochondrial fusion and architecture (A). Isolated proteins from spermatozoa of undisturbed and stressed rats were used for immunoprecipitation analysis with MFN2 antibody, followed by Western blot for PRKAc and p38 MAPK (B). The representative blots are shown as panels. Data from scanning densitometry were normalized on MFN2 (internal control). Values are shown as bars above the photos of blots. Data are presented as green dots connected with a green line or green bars for the control group, and blue squares connected with a blue line or blue bars for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, # vs. the control group of ZT3 time point.

Figure 5

Transcription of mitochondrial fission markers is significantly changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of mitochondrial fission. Data are presented as green dots connected a with green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, # vs. the control group of ZT3 time point.

Figure 6

Transcription of mitochondrial autophagy markers is significantly changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of mitochondrial autophagy. Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, ^# vs. the control group of ZT3 time point.

Figure 7

Transcription of mitochondrial functionality markers is significantly changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of mitochondrial functionality. Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, ^# vs. the control group of ZT3 time point.

Figure 8

Transcription of markers of cAMP signaling regulating mitochondrial dynamics and functionality as well as spermatozoa number and functionality is changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of the cAMP signaling pathway. PCA of markers of the cAMP signaling pathway on active/inactive phase; Dim1 and Dim2 represent the first two PCs and % of the retained variation. Cos2 estimates the qualitative representation of variables ( Supplementary Table S2 ). Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, # vs. the control group of ZT3 time point.

Figure 9

Transcription of markers of MAPK signaling regulating mitochondrial dynamics and functionality as well as spermatozoa number and functionality is changed in spermatozoa of acutely stressed adult rats in a time-dependent manner. Isolated RNA from spermatozoa of undisturbed and stressed rats was used for the analysis of the transcriptional profile of markers of the MAPK pathway. PCA of markers of the MAPK signaling pathway on active/inactive phase; Dim1 and Dim2 represent the first two PCs and % of the retained variation. Cos2 estimates the qualitative representation of variables ( Supplementary Table S3 ). Data are presented as green dots connected with a green line for the control group, and blue squares connected with a blue line for the 1x3hIMO group, and are mean ± SEM values of two independent in vivo experiments. Statistical significance was set at p < 0.05: * vs. the control group of the same time point, # vs. the control group of ZT3 time point.

Figure 10

Heat map analysis of the transcriptional profile of the mitochondrial dynamic and functionality markers (A) and the signaling molecules regulating mitochondrial dynamics and functionality (B) in spermatozoa of acute stressed adult rats. Heat map analysis showing different patterns of transcription at different time points in spermatozoa after the acute immobilization stress. The relative fold change in gene expression for the aforementioned genes was compared in different time points (ZT3, ZT11, ZT17, and ZT23). Color from red to green indicates low to high expression.

Figure 11

The transcription pattern in spermatozoa of acutely stressed adult rats with different recovery periods (ZT3, ZT11, ZT17, and ZT23). Data shown represent the transcriptional pattern of the genes for mitochondrial dynamics/functionality markers (A) as well as cAMP and MAPK signaling pathway-related molecules (B). Points represent a deviation in the transcription of a particular gene at different ZT time points.

Figure 12

PCA of mitochondrial dynamics (A), cAMP signaling pathway (B), and MAPK signaling pathway (C) gene expression on active/inactive phase; Dim1 and Dim2 represent the first two PCs and % of the retained variation. Cos2 estimates the qualitative representation of variables ( Supplementary Tables S1–S3 ).

Figure 13

Acute stress, the most common stress in human society, significantly changes 91% of followed mitochondrial dynamics and functionality markers as well as 91% of signaling molecules regulating spermatozoa homeostasis and mitochondrial dynamics/functionality. The most prominent changes were observed 20 h after the end of the stress. The physiological significances are the recovery of spermatozoa number and functionality (positive acrosome reaction). Furthermore, the interactions between the proteins important for spermatozoa homeostasis and functionality (MFN2 and PRKA catalytic subunit, MFN2, and p38MAPK) are shown for the first time.