Ethics Statement

All experiments were approved by the Local Ethics Committee for Experimentation on Animals no. 1 (Warsaw, Poland) and were performed in compliance with the national regulations.

Animals and reagents

Young (2–4 months old) and old (13–16 months old) outbred Tar:SWISS (SWISS), inbred C57BL/6Tar (C57BL/6), and hybrid F1 (C57BL/6Tar × CBA/Tar) mouse females and hybrid F1 (C57BL/6Tar × CBA/Tar) mouse males (4–6 months old) were maintained in the Animal Facility of the Faculty of Biology, University of Warsaw at 14:10 light/dark cycle and provided with food (standard chow diet; Labofeed H Standard (Wytwórnia Pasz “Morawski”, Poland)) and water ad libitum. Males and females were kept in the same room, in open cages with at least 60–100 cm² of cage space per mouse, depending on the mouse weight. C57BL/6Tar inbred substrain was derived from B6J substrain and CBA/Tar—from CBA/W substrain; both were bred separately for 20 generations. Old C57BL/6 females were used before the experiments as breeders. Animals were sacrificed by cervical dislocation. All reagents were purchased from Sigma-Aldrich Merck unless stated otherwise.

Oocyte collection

To obtain GV oocytes (i.e., oocytes in prophase of the 1st meiotic division), female mice were primed with an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG, Intervet). GV oocytes were isolated 48 h later from ovaries into M2 medium (M16 buffered with Hepes [33]) and cultured for 16 h (in vitro maturation) in M16 medium. To obtain ovulated oocytes (in metaphase of the 2nd meiotic division, MII), the PMSG injections were followed 48 h later by 10 IU of human chorionic gonadotrophin (hCG, Intervet). Ovulated oocytes were recovered from oviducts 15 h after hCG and placed in hyaluronidase solution (150 IU/ml in phosphate-buffered saline, PBS) to remove the cumulus cells. Denuded oocytes were washed in M2 medium.

Sperm collection

Epididymal sperm for all experiments involving in vitro fertilization was isolated from F1 males and capacitated in 0.5 mL of fertilization medium with 5 mg/ml bovine serum albumin (BSA) [34] for 1.5–2 h in 37.5 °C and 5% CO₂ in the air.

Imaging of Ca²⁺ oscillations

Oocytes loaded with 5 µM fluorescent Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM (Molecular Probes, Thermo Fisher Scientific; in M2 medium, 30 min) were subjected to acidic Tyrode solution (pH 2.5) [35] to remove zonae pellucidae. Oocytes without zonae were transferred to a glass-bottom dish (MatTek Corporations) with 2 ml of M2 medium without BSA and allowed to stick to the glass bottom. Next, the dish was placed on a time-lapse imaging system (Zeiss Axiovert microscope with an AxioCam HRm camera) equipped with an environmental chamber sustaining a temperature of 37.5 °C. 1–2 µl of capacitated sperm suspension (approximate concentration of 2 × 10⁷ spermatozoa/ml) were added to the oocytes. Time-lapse imaging was initiated ∼17 h post hCG; single-plane images were taken every 10 s for ∼7 h. Oocytes were illuminated with light passing through a 450–490 nm excitation filter, and the emitted light was collected with a 500–550 nm emission filter (exposure time 50 ms, 4 × 4 binning). Oocytes of each type (defined by the age and female strain) were imaged separately.

Changes in cytoplasmic Ca²⁺ concentration were assessed by measuring the mean intensity of Oregon Green BAPTA fluorescence in time. The Fiji software ( https://imagej.net/Fiji) was used for the analysis. To avoid additional variability between the experiments caused by the different extent of dye loading ( Supplementary Figure S1A), the initial (pre-fertilization) mean intensity of fluorescence was calculated for each oocyte and then used to normalize the measurements in this oocyte. The resulting values are ratios: measured fluorescence intensity (F)/initial fluorescence intensity (F₀). The rates of increase and decrease of Ca²⁺ concentration during the Ca²+ transients were calculated as tangents of the rising/decreasing slopes of the Ca²⁺ transients (“a” parameter in y = ax + b linear function fitted into these slopes). The recordings were also analyzed for the timing of pronuclei formation (Oregon Green BAPTA dye does not accumulate in pronuclei, so they are visible as darker circular regions in the cell). To avoid the confounding effects of photobleaching and the dye compartmentalization that may appear during prolonged imaging, we analyzed only oocytes fertilized within the first 2 h of the recording, and we measured amplitudes only of the 1st and 3rd Ca²⁺ transients ( Supplementary Figure S1B). Only monospermic oocytes were included in the final analysis and the number of fused sperm was assessed based on the number of fertilization cones and/or pronuclei formed. The numbers of analyzed oocytes and experimental repetitions are included in  Supplementary Table S1.

Table 1.

Ovulation yields in young and old females of different genetic backgrounds.

Ca²⁺ store measurement

Oocytes were loaded with 5 µM Oregon Green BAPTA-1 AM (in M2 medium, 30 min) and transferred to a glass-bottom dish with M2 medium devoid of Ca²⁺ and Mg²⁺ ions. After 2 minutes of time-lapse imaging thapsigargin (TG) or A23187 ionophore were added to the medium to the final concentration of 10 µM. Single-plane images were taken every 10 s for ∼30 min (the settings were the same as described for imaging of Ca²⁺ oscillations). Oocytes of each type (defined by the age and female strain) were imaged separately.

Changes in the Oregon Green BAPTA fluorescence were analyzed as described for the imaging of Ca²⁺ oscillations. The amplitude of the Ca²⁺ transient (a difference between the maximum fluorescence intensity and the fluorescence intensity at the beginning of the Ca²+ increase) and the area under the curve (AUC) of the Ca²⁺ transient (calculated for the first 10 min following the onset of Ca²⁺ increase relative to baseline using the trapezoidal method; modified from [36]) were measured. All oocytes included in the analysis were viable at the end of the imaging period. The numbers of analyzed oocytes and experimental repetitions are included in Table 2.

Immunofluorescence staining

Oocytes were fixed for 30 min in room temperature (RT) in 4% formaldehyde solution prepared in PBS, permeabilized with (1) 0.2% Triton-X100 in PBS (25 min, RT) and blocked with 3% BSA in PBS for ITPR1 and calnexin stainings or (2) 0.5% Triton-X100 in PBS (25 min, RT) and blocked with 10% fetal bovine serum (FBS) solution prepared in PBS for ATP2A2 staining. Calnexin was labeled with a rabbit polyclonal antibody (Abcam, cat. no. ab22595; diluted 1:200 in 3% BSA) and ITPR1 with a rabbit polyclonal antibody (Abcam, cat. no. ab5908; diluted 1:100 in 3% BSA), both followed by an Alexa Fluor 633-conjugated goat anti-rabbit IgG (Invitrogen, Thermo Fisher Scientific, cat. no. A21071; diluted 1:200 in 3% BSA). ATP2A2 was labeled with a mouse monoclonal antibody (Abcam, cat. no. ab2861; diluted 1:100 in 10% FBS) followed by a TRITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., cat. no. 115–025-068; diluted 1:200 in 10% FBS). Embryos were incubated in the primary antibodies overnight at 4 °C, washed in PBS and 3% BSA or 10% FBS, and then, incubated with the secondary antibody for 2 h in RT. Old and young oocytes were analyzed on an inverted confocal microscope (Zeiss and Olympus) using the same imaging settings. Mean fluorescence intensity was measured for the Z-stack projections prepared in the average intensity mode. The Fiji software was used for the analysis.

Western blotting

Expression of ITPR1 protein was examined in samples of 70–80 oocytes, depending on the experiment. Cell lysates were mixed with 4× NuPage LDS sample Buffer and 10× NuPage Sample Reducing Agent (Invitrogen, Thermo Fisher Scientific) and heated for 10 min in 70 °C. The samples were subjected to NuPage Novex 3–8% Tris-Acetate gels (Invitrogen, Thermo Fisher Scientific), and separated proteins were transferred onto PVDF membranes (Hyperbond-P, Amersham Biosciences). The blots were then stained with Ponceau S to confirm the equal sample load, blocked in 5% non-fat milk solution prepared in Tris-buffered saline with Tween20 (TTBS), and probed for 1 h with a rabbit polyclonal antibody (Rbt03) raised against a 15 amino acid peptide sequence of the C-terminal end of the ITPR1 [37] diluted 1:350 in 5% non-fat milk in TTBS. A goat anti-rabbit antibody (Bio-Rad) conjugated with horseradish peroxidase diluted 1:5000 was used as the secondary antibody in 1-h-long incubation. Detection was performed by the enhanced chemiluminescence technique using SuperSignal West Dura Extended Duration Substrate reagents (Pierce, Thermo Fisher Scientific) according to the manufacturer's instruction.

RT-qPCR

Oocytes were transferred in groups of 15–20 (depending on the experiment) into 20 µl of Lysis/Binding Buffer (Dynabeads mRNA DIRECT Micro Kit, Thermo Fisher Scientific) and stored in –80 °C until further analysis. mRNA was isolated from the samples using the Dynabeads mRNA DIRECT Micro Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. In short, thawed samples were rotated with 10 µl of paramagnetic oligo(dT)₂₅ bead suspension for 30 min at RT. mRNA was eluted from the beads by adding 10 µl of DEPC-treated water and heated for 10 min at 70 °C with 0.5 µg oligo(dT)_12–18. The reverse transcription was performed in a total volume of 20 µl using 200 U of Superscript II Reverse Transcriptase, 0.5 mM dNTPs, and 40 IU RNase inhibitor (Invitrogen, Thermo Fisher Scientific) at 42 °C for 50 min. Synthesized cDNA was diluted twice (to 40 µl) with nuclease-free water (Thermo Fisher Scientific) and subjected (3 µl per sample) to real-time PCR using TaqMan Gene Expression MasterMix and TaqMan Gene Expression Assays probes (Itpr1/Ip3r1: cat. no. Mm00439907_m1; Atp2a2/Serca2: Mm01275320_m1; Erp44/Txndc4: Mm00466483_m1; Pdia3/Erp57: Mm00433130_m; Actb: Mm01205647_g1; Applied Biosystems, Thermo Fisher Scientific) in StepOne Real-Time PCR System thermocycler (Applied Biosystems, Thermo Fisher Scientific; 50 °C/2 min; 60 °C/10 min; 50 cycles: 95 °C/15 s, 60 °C/1 min). The relative level of expression was calculated using the 2–△C^t method [38] with Actb expression used for normalization.

Statistical analysis

Normality of the data distribution was verified with the Shapiro–Wilk test. Data with a normal distribution were analyzed with the Student's t-test. When the distribution was not normal, we applied the Mann–Whitney U test. The differences between groups were considered statistically significant for P < 0.05. Spearman's rank correlation coefficient was used to show correlations between analyzed numerical variables.

Figure 1.

Effect of maternal aging on the pattern of fertilization-induced Ca²⁺ oscillations in oocytes from F1, SWISS, and C57BL/6 mice. (A) Summary of the alterations induced by maternal aging in Ca²⁺ oscillations in fertilized oocytes. Red marks values lower, gray—the same, and green—higher in old oocytes than in their young counterparts. The amounts of Ca²⁺ in the ER and the whole cell correspond to the AUCs for the TG- and A23187-induced Ca²⁺ increases, respectively. (B) The number of Ca²⁺ transients in young (n = 57 F1, 93 SWISS, 36 C57BL/6) and old (n = 30 F1, 74 SWISS, 17 C57BL/6) oocytes. (C) Mean interval between two consecutive Ca²⁺ transients calculated for the first 2 h of Ca²⁺ oscillations in young (n = 57 F1, 92 SWISS, 36 C57BL/6) and old (n = 29 F1, 74 SWISS, 17 C57BL/6) oocytes. (D) The total duration of Ca²⁺ oscillations in young (n = 57 F1, 92 SWISS, 36 C57BL/6) and old (n = 29 F1, 74 SWISS, 17 C57BL/6) oocytes. (E) Duration and (F) amplitude of the 1st Ca²⁺ transient in young (n = 55 F1, 92 SWISS, 36 C57BL/6) and old (n = 27 F1, 73 SWISS, 18 C57BL/6) oocytes. (G) Rate of Ca²⁺ increase during the 1st Ca²⁺ transient in young (n = 54 F1, 92 SWISS, 36 C57BL/6) and old (n = 27 F1, 74 SWISS, 18 C57BL/6) oocytes. (H) Rate Figure 1. (continued) of Ca²⁺ decrease during the 1st Ca²⁺ transient in young (n = 56 F1, 92 SWISS, 36 C57BL/6) and old (n = 30 F1, 74 SWISS, 16 C57BL/6) oocytes. (B–H) Graphs present medians and the 1st and the 3rd quartile values. The ends of the whiskers are set at 1.5*IQR above the third quartile and 1.5*IQR below the first quartile. Dots show the minimum and maximum values if they are outside the range (outliers).

Maternal aging decreased ovulation yields in all examined genetic backgrounds

To examine the effects of maternal aging on oocytes' ability to generate correct Ca²⁺ response to fertilization, we needed to isolate MII oocytes. We noticed that reproductively aged females (13–16 months old, an equivalent of 36–45 years of age in humans [39]) of all three examined strains/crosses ovulated significantly fewer MII oocytes than their young counterparts (Table 1). This observation accords with the previously published data regarding the impact of maternal aging on mouse female fertility (e.g., [3, 19, 40–44]) and confirms that mice used in our experimental set-up indeed were in advanced reproductive age.

Maternal aging affects the pattern of Ca²⁺ oscillations in a strain-dependent manner

To investigate how advanced reproductive age affects the pattern of fertilization-induced Ca²⁺ oscillations, we labeled MII oocytes recovered from young and old F1, SWISS, and C57BL/6 females with Oregon Green 488 BAPTA-1 AM, a f luorescent Ca²⁺ indicator, fertilized them in vitro with F1 spermatozoa, and subjected them to time-lapse imaging. Next, we analyzed f luorescence intensity that ref lected Ca²⁺ concentration in ooplasm and calculated several parameters describing the pattern of sperm-triggered Ca²⁺ oscillations, such as the number of Ca²⁺ transients, the mean interval between subsequent Ca²⁺ transients (the frequency of Ca²⁺ oscillations gradually decreases over time, so we calculated it only for the first 2 h after fertilization), the total duration of Ca²⁺ oscillations, the duration and amplitude of Ca²⁺ transients (we analyzed the 1st and the 3rd Ca²⁺ transients as examples), and the rate of increase/decrease of Ca²⁺ during the Ca²⁺ transients. As the number of fused sperm affects the pattern of Ca²⁺ oscillations [45], we included in our analysis only oocytes fertilized by a single sperm.

We showed that the pattern of Ca²⁺ oscillations was altered more severely in oocytes from old F1 and C57BL/6 mice than old SWISS mice. Maternal aging decreased the number and frequency of Ca²+ transients in F1 oocytes. However, the duration of Ca²⁺ transients (at least the 1st and the 3rd) was longer, and the amplitude and the rate of rise of the 3rd Ca²⁺ transient were higher in oocytes obtained from aged F1 females. In old C57BL/6 oocytes, Ca²⁺ oscillations lasted for shorter and consisted of fewer Ca²⁺ transients than in young oocytes. Moreover, the 1st Ca²⁺ peak in aged oocytes lasted on average for longer, and rates of its increase and decrease were slower than in young oocytes. SWISS oocytes were affected by the advanced maternal age to the lowest extent: in oocytes from old females, only the duration of the 1st and the 3rd Ca²⁺ transients was shorter than in oocytes from young females (Figure 1,  Supplementary Table S1,  Supplementary Figures S2 and  S3).

It is important to note that our measurements of the amplitudes and, in consequence, also the rates of Ca²⁺ increase/decrease, are only estimates, as Oregon Green BAPTA is not a ratiometric dye, and its response to increasing Ca²⁺ concentration is not linear (its output flattens for higher Ca²⁺ concentrations) [46]. Therefore, we may have less ability to detect small differences in the amplitudes within the upper part of the dye working range. In summary, maternal aging alters the pattern of Ca²⁺ oscillations in fertilized oocytes in a different manner depending on the genetic background of the cells: F1 and C57BL/6 oocytes are more and SWISS oocytes less affected by the female advanced reproductive age.

Cellular Ca²⁺ store changes during maternal aging in a strain-dependent manner

It is possible that strain-dependent differences in the impact of maternal aging on the pattern of Ca²⁺ oscillations in fertilized oocytes are a consequence of different age-related alterations to the oocyte's Ca²⁺ store. Therefore, we examined whether the effect of maternal aging on the amount of Ca²⁺ stored in oocytes differed as well between the mouse strains. To this end, we labeled oocytes with Oregon Green 488 BAPTA-1 AM and treated them with thapsigargin (TG) or A23187 ionophore to assess Ca²⁺ amount stored in either ER cisterns or the whole cell, respectively. TG blocks ATP2A2 pump in the ER membrane and thus allows for a gradual leakage of Ca²+ from ER cisterns into the cytoplasm [47, 48]. On the other hand, A23187 ionophore is an ion-carrier that forms stable complexes with divalent cations and transports them across lipid bilayers such as cellular membranes [49]. Ca²⁺ increase induced by the reagents was recorded by time-lapse imaging, fluorescence intensity was analyzed, and amplitudes and areas under the curve (AUCs) for the Ca²+ transients were calculated.

We showed that maternal aging decreased the amount of Ca²+ accumulated in the ER, as assessed by the Ca²⁺ spike amplitude and AUC, in both F1 and C57BL/6 oocytes (Table 2, Figure 2A and C). It accords with our earlier observation that old F1 and C57BL/6 oocytes generated fewer Ca²⁺ transients in response to fertilization than their young counterparts and, additionally, that in old C57BL/6 oocytes Ca²⁺ oscillations lasted for shorter and in old F1 oocytes—were less frequent than in young oocytes (Figure 1B–D,  Supplementary Table S1). The amplitude of TG-induced Ca²⁺ transient was increased, whereas the AUC was decreased in old SWISS oocytes comparing to their young counterparts (Table 2, Figure 2B). However, as the dynamics of Ca²⁺ response to TG was significantly different in young and old SWISS oocytes, the AUC of the Ca²+ transient is a better measure of the ER Ca²⁺ store than the Ca²+ transient amplitude. The slower Ca²⁺ response to TG observed for young SWISS oocytes suggests either lower responsiveness of ATP2A2 to the drug or decreased Ca²⁺ leak from the ER.

On the other hand, the amount of Ca²⁺ stored in the whole cell, as assessed by the amplitude and AUC of the Ca²⁺ response to A23187, was higher in oocytes from old F1 females and lower in oocytes from old C57BL/6 than in oocytes from their young counterparts (Table 2, Figure 2A and C). The amplitude of ionophore-induced Ca²⁺ transient was lower in old than in young SWISS oocytes, but the AUC remained the same in both types of oocytes. This suggests that if there is any decrease in the amount of Ca²⁺ stored in the aged SWISS oocytes, it is relatively small (Table 2, Figure 2B). In summary, our data indicate that maternal aging decreases the ER Ca²⁺ store but, depending on the genetic background, may differently impact the amount of Ca²⁺ stored in other organelles (e.g., mitochondria).

Figure 2.

Effect of maternal aging on the amount of Ca²⁺ stored in oocytes from F1, SWISS and C57BL/6 mice. (A–C) Mean Ca²⁺ release triggered by thapsigargin (TG), calculated for young (in red; n = 52 F1, 48 SWISS, 44 C57BL/6) and old (in black; n = 26 F1, 30 SWISS, 25 C57BL/6) oocytes isolated from (A) F1, (B) SWISS, and (C) C57BL/6 mice. (D and E) Mean Ca²⁺ release triggered by A23187 ionophore, calculated for young (in red; n = 45 F1, 52 SWISS, 50 C57BL/6) and old (in black; n = 35 F1, 50 SWISS, 28 C57BL/6) oocytes isolated from (D) F1, (E) SWISS, and (F) C57BL/6 mice. Mean values ±SD are shown. Recordings were synchronized according to the time-point when the cytoplasmic Ca²⁺ concentration in oocytes started to rise.

Table 2.

Amount of Ca²⁺ stored in oocytes obtained from young and old females of different genetic backgrounds.

Maternal aging affects the expression of Ca²+ homeostasis genes in a strain-depending manner but does not alter the distribution of ER cisterns

Alterations in the pattern of Ca²⁺ oscillations observed in oocytes obtained from aged females may be caused by changed expression of proteins involved in Ca²⁺ homeostasis. Pan and co-workers [3] and Grøndahl and co-workers [4] showed in their microarray analyses that maternal aging affects the expression of inositol 1,4,5-trisphosphate receptor 1 (Itpr1), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (Atp2a2), Endoplasmic reticulum resident protein 44 (Erp44), and Protein disulfide-isomerase A3 (Pdia3) genes in mice and humans, thus we decided to investigate the expression of these genes on mRNA and protein level in our experimental set-up. ITPR1 and ATP2A2 proteins regulate the flow of Ca²⁺ ions between ER cisterns and cytoplasm, whereas ERp44 and PDIA3 regulate ITPR1 and ATP2A2 activities, respectively (reviewed in [50]). Messenger RNA expression was assessed in ovulated MII oocytes, but because ovulation yield was very low in aged females, in the experiments detecting proteins we used GV oocytes isolated from ovaries and matured in vitro. To analyze mRNA expression, we isolate mRNAs using oligo (dT)₂₅-coated paramagnetic beads. The method is very effective for transcripts with poly(A) tails longer than 25 nts [51], but transcripts with shorter poly(A) tails might have been underrepresented in our analysis. The majority of mRNAs present in mammalian cells have on average longer poly(A) sequences (50–250 nts), but indeed, in oocytes, some maternal mRNAs stored for future translation may have short poly(A) tails; such tails usually mark mRNAs that are translationally dormant; therefore, even if present, they do not participate in the protein production at the given time-point (reviewed in [52, 53]).

The qPCR analysis indicated that in SWISS oocytes maternal aging did not change mRNA levels for any of the examined genes. In F1 oocytes, we noticed a decrease in mRNA expression of Itpr1 and Atp2a2. However, it was not detectable on the protein level; on contrary, the ITPR and ATP2A2 expression seemed to be slightly higher in aged F1 oocytes (as assessed by immunofluorescence staining for ITPR1 and ATP2A2 and by Western blot for ITPR1; we did not manage to detect ATP2A2 signal in immunoblotting) (Figure 3A–F,  Supplementary Figure S4). The cytoplasmic distribution of ITPR1 and ATP2A2 observed by us in the immunostainings accords with the data published previously [54, 55] and mirrors the distribution of calnexin, an ER marker (Figure 3E and G). Interestingly, we did not observe an upshift in the position of the ITPR1 band in samples from old vs. samples from young oocytes, which indicates that maternal aging did not lead to abundant phosphorylation of the ITPR1 protein [56, 57] (Figure 3F). In oocytes obtained from C57BL/6 females, maternal aging led to a decline in the expression of Erp44 mRNA. Unfortunately, we were able to detect ERp44 protein neither by immunostaining nor by Western blot, so we cannot verify whether the aging impacts the protein amount (Figure 3A–F).

The dynamics of Ca²⁺ oscillations may also depend on the distribution of the ER cisterns [56–58]. Therefore, we examined whether maternal aging changes the distribution of the ER network in MII oocytes. As before, we used MII oocytes that matured in vitro, and we labeled them for calnexin, an ER marker. Our analysis indicated that ER cisterns were distributed as previously shown [56, 57, 59] and that maternal aging did not affect their localization, regardless of the female's genetic background (Figure 3G).

Figure 3.

Distribution of ER cisterns and expression of genes involved in Ca²⁺ homeostasis in oocytes from young and old F1, SWISS, and C57BL/6 mice. (A–D) Relative expression of mRNA for (A) Itpr1, (B) Atp2a2, (C) Erp44, and (D) Pdia3 genes in young and old oocytes. PCR analysis was repeated six times. (E) Representative images of immunofluorescence stainings for ITPR1 and ATP2A2 in F1 young (n = 49 and 29, respectively) and old (n = 13 and 28, respectively) oocytes. Regions marked with dashed line are magnified in the inserts. Scale bar 20 µm. (F) Western blot analysis for ITPR1 in F1 young and old oocytes. Numbers below the blots present mean results (±SD) of the densitometric analysis from three experiments. Ponceau S staining was used to confirm an equal sample loading. (F) Representative images of immunofluorescence stainings for calnexin, the ER marker, in young (n = 29 F1, 18 SWISS, 12 C57BL/6) and old (n = 34 F1, 20 SWISS, 8 C57BL/6) oocytes.

Taken together, even though we detected some age-related differences in mRNA expression of genes involved in Ca²⁺ homeostasis, those differences did not necessarily translate directly to protein levels. Moreover, maternal aging did not affect the distribution of ER cisterns. Consequently, neither of those parameters can fully explain the age-dependent change in the pattern of Ca²⁺ oscillations.

Maternal aging alters the timing of pronuclei formation in a strain-dependent manner

As the pattern of Ca²⁺ oscillations regulates early embryonic cell cycle events [9, 10], we wished to examine whether age-related modifications in Ca²⁺ oscillations coincide with altered timing of pronuclei formation in zygotes. To this end, we measured the time between the initiation of the first Ca²⁺ transient and the first appearance of pronuclei in zygotes obtained from young and aged F1, SWISS, and C57BL/6 females. We observed that pronuclei formed later in zygotes from old F1 and SWISS females than in embryos from their young counterparts, whereas in CB57BL/6 zygotes we noticed the opposite trend (Figure 4A). It has been shown that the events accompanying oocyte activation rely on the total duration of the period when the cytoplasmic Ca²⁺ concentration is increased [9, 10]. Therefore, we examined whether the timing of pronuclei formation correlates with the number of Ca²⁺ transients and the mean period between subsequent Ca²⁺ transients (translating to the frequency of Ca²⁺ oscillations), as these parameters reflect the total time when the cytoplasmic Ca²⁺ concentration is elevated. Old and young oocytes were analyzed together and a significant positive correlation was observed only between the timing of pronuclei formation and the mean period between subsequent Ca²⁺ transients for C57BL/6 and SWISS strains (Figure 4B). Therefore, our results suggest that the frequency of sperm-induced Ca²⁺ oscillations may affect the timing of pronuclei formation at least in some mouse strains. However, as the frequency of Ca²⁺ peaks did not actually differ between young and old oocytes from C57BL/6 and SWISS mice, there must be another factor(s) that explains the different rates of interphase onset in embryos obtained from young and old females.