Oocyte aging is controlled by mitogen-activated protein kinase signaling

Abstract

Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions already during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double-strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK-1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK-1, oocyte quality deteriorates more rapidly than in wild-type worms, whereas reduction of MPK-1 levels enhances quality. Thus, our data suggest that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline.

Keywords: C. elegans; MAPK; aging; fertility; meiosis; oocyte; oogenesis.

FIGURE 1

Aging leads to reduced number of germ cells. (a–e) Images of DAPI‐stained whole mount gonads from worms at the indicated ages. The different oogonial stages are marked. Insets show regions containing LZ nuclei (1) and mature oocytes (2). Two categories of day 10 gonads are shown: organized and disorganized. Arrowheads—endomitotic nuclei. Dashed circle in (e) indicates a nucleus with diakinesis morphology located distally. Scale bar = 50 µM. (f–k) Box plots depict the average number (x) and the individual measurements (circles) of (f) total nuclei in the gonads and (g–k) specific stages per gonad from worms at the indicated ages. Mann–Whitney p value: NS—not significant, *<0.05, **<0.01, ***<0.001, and ****<0.0001. n values are marked below each box

FIGURE 2

Changes in meiotic processes in aged germlines. (a) Average number of CED‐1::GFP apoptotic nuclei per gonad from worms at the indicated ages. n ≥ 7 gonads. Mann–Whitney p value: NS—not significant, *<0.05. (b) RAD‐51 (red) and DAPI (blue) staining of whole mount gonad arms at the indicated ages. Scale bar = 50 µM. Asterisk indicates the distal side of the gonad. (c) Average number of RAD‐51 foci per nucleus at the different oogonial stages. (d) COSA‐1::GFP (green) and DAPI (blue) staining of late pachytene nuclei at the indicated ages. Scale bar = 10 µM. (e) Histogram of the relative percentages of nuclei with four, five, and six COSA‐1 foci per late pachytene nucleus at the different ages. n = 200 nuclei (f) Average number of RAD‐51 foci per gonad nuclei at different pachytene stages in wild‐type day 4 gonads of control and mpk‐1 RNAi worms. n ≥ 80 gonads. Mann–Whitney p value: NS—not significant, *<0.05, ***<0.001, and ****<0.0001

FIGURE 3

Homologs are located at greater distance from each other in old worms. (a) The z‐projected images of DAPI‐stained bivalents in mature oocytes at day 1 and day 10. Scale bar = 1 µM. (b) 2D chromatin density maps obtained by projecting the z‐stacks in panel (a) for day 1 and day 10 bivalents from mature oocytes. Chromatin density is indicated both by color and surface height. The 2D distance between the projected Gaussian centers is defined as L. The difference in fluorescence between the minimum value along L and the mean value of the two Gaussians centers is defined as H. (c) Box and whisker plot of the distance L. n = 20. (d) Box and whisker plot of the fluorescence difference H. n = 20. (e). Box and whisker plot of the Jensen–Shannon distance between the 3D Gaussian distributions of the two homologs in each pair. n = 20. (f) Images of bivalents from day 1 and day 8 mature oocytes stained for LAB‐1 (red), COSA‐1::GFP (green), and DAPI (blue). n = 32

FIGURE 4

Oocyte get smaller with age. (a and b) Proximal end of gonad arms at the indicated ages (a) stained with DAPI and imaged with fluorescence microscopy and (b) imaged with DIC. Arrows indicate pairs of diakinesis nuclei in the same row. White dashed lines indicate the circumference of the mid plane of oocytes. Scale bar =20 µM. (c) Average area of −1 oocytes at the different ages. n=20 oocytes for day 1, n = 30 for day 4, and n = 11 for day 8. (d) Proximal end of gonad arms of rrf‐1 day 4 gonads of control and mpk‐1 RNAi worms. White dashed lines indicate the circumference of the mid plane of oocytes. Scale bar =20 µM. (e) Average area of −1 oocytes. N ≥ 13 gonads. Mann–Whitney p value: NS—not significant, *<0.05, and ***<0.001

FIGURE 5

MPK‐1 activation in the gonad changes during aging. Images of whole mount gonads from worms of the indicated ages stained for dpMPK‐1 (red) and DAPI (blue). Average levels of dpMPK‐1 signal are indicated below the images. n = 8 gonads for day 1, n = 8 for day 4, n = 5 for day 8, and n = 10 for day 10

FIGURE 6

Viability of aged oocytes can be attenuated by MPK‐1 activation. (a) Illustration of the experimental design. (b–e) Average embryonic viability of the first ~10 fertilized oocytes after the arrest in (b). mutants that lead to over activation of MPK‐1 in oocytes, (c) in wild‐type and mpk‐1(ga111) at semi‐permissive (20 °C) temperature, (d). in wild‐type and rrf‐1 worms depleted of mpk‐1 using RNAi, and (e). in worms that were kept in the presence an MPK‐1 inhibitor during the arrest. Mann–Whitney p value: **<0.01 and ****<0.01. n values are indicated on the columns