Maternal aging reduces female fecundity and alters offspring phenotype in a sex-specific manner

Fecundity assessments

Female fecundity across the reproductive lifespan was analysed from litter size and pup weaning data collected from breeder pairs in a mouse colony, located at and managed by the Laboratory Animal Services, The University of Adelaide. Breeder pairs (one male and one female) were housed together at ~2.5 months of age and until a maximum of 18 months of age. Data was collected on number of litters, litter size at birth and number of pups weaned 3 weeks post-birth.

Offspring generation and body weight measurement

Offspring were produced by mating either young or reproductively aged female mice to young male mice. For this, young (8-week-old, nulliparous) and reproductively aged (40-week-old multiparous (i.e. ‘ex-breeder’)) C57BL/6J female mice, were obtained from the Animal Resources Centre (Canning Vale, WA, Australia). Female mice were housed in groups of five to seven until the females were 52 weeks of age (12 months old, equivalent to 41 years of age in women (Dutta and Sengupta 2016)), and the young controls were 12 weeks old (3 months old). Male mice were C57BL/6J at 8–12 weeks old and proven fertile. Females were housed with a male (1:1) for 1 week, separated, and then weighed 1 week later; with pregnancy indicated by a ≥ 3 g increase in body weight. After birth, the offspring remained with the dam until weaning at 3 weeks of age. Upon weaning, offspring were individually identified and housed in groups of five to seven with offspring from other mothers of the same type. Offspring body weights were measured weekly on week 3–10 postnatal, and then at 12 and 16 weeks postnatal. At 12 weeks of age, the offspring underwent behaviour testing. Glucose tolerance tests (GTT), and insulin tolerance tests (ITT), were conducted 2 weeks later and tissues were collected at 16 weeks of age.

Glucose and insulin tolerance test

Prior to the glucose tolerance test (GTT) at 13–14 weeks old, the offspring were fasted for 5–6 h, then injected intraperitoneally with 2 g glucose per kg of body weight in 0.9% NaCl solution. The same animals underwent insulin tolerance test (ITT) 1 week later at 14–15 weeks old. Mice received an intraperitoneal injection of 0.75 IU of Actrapid® Penfill® (Novo Nordisk Pharmaceuticals Pty Ltd., North Sydney, NSW, Australia) per kg of body weight in 0.9% NaCl solution. For both GTT and ITT, blood glucose was measured in tail tip blood samples at 0, 15, 30, 60 and 120 min post challenge using Accu-check Performa glucose meter (Roche Diabetes Care, North Ryde, NSW, Australia).

Blood collection and organ weight measurement

Tissues were collected from the offspring at 16 weeks old. For this, mice were fasted for 3 h, then blood was collected from a subset of animals by cardiac puncture for immune cell analysis. Briefly, mice were anaesthetised with 125–250 mg/kg 2,2,2, tribromoethanol (Sigma-Aldrich Pty Ltd, North Ryde BC, NSW, Australia) via intraperitoneal injection; and approximately 500 μL blood was collected from the heart using an 18G needle connected to a heparinised (10 USP unit/mL) syringe. The heparin-blood mixture was gently mixed and immediately used for immune cell analysis. Mice were humanely killed by cervical dislocation followed by whole body weight and organ measurement, namely: abdominal fat pad, spleen, liver, kidneys, heart and reproductive organs (testis or uterus plus ovaries).

Immune cell staining and flow cytometry

Reagents for immunophenotyping assays were acquired from Becton Dickinson Pty Ltd. (see Supplementary Table S1). 200 μL of heparinised blood was transferred into a 1.7 mL Eppendorf tube containing antibody cocktail (see Table S2), mixed and incubated at 4°C for 30 min. Samples were then transferred into 15 mL tubes and red blood cells (RBC) were lysed by adding 5 mL RBC lysis buffer (150 mM NH₄Cl, 10 mM NaHCO₃ and 1 mM EDTA), mixing and incubation at room temperature for 10 min. The sample was then centrifuged at 300g at room temperature for 5 min, the supernatant discarded, and the RBC lysis process repeated. After the second centrifugation, the cells were resuspended with 100 μL 0.5% bovine serum albumin (BSA)-supplemented phosphate buffered saline and transferred into a 5 mL test tube. The sample was analysed by flow cytometry and the proportion of immune cell populations were quantified using LSRFortessa X-20 Cell Analyser and FACSDiva analysis software (BD Biosciences, San Diego, CA, USA). The gating strategy for immune cell types was based on previous established methods (Hensel et al. 2019), and is shown in Supplementary Fig. S1. The specific immune cell proportion is calculated from number of each specific immune cell type divided by total number of cells multiplied by 100. Validation using blood samples taken from young and old mothers showed that the gating strategy employed successfully quantified the proportion of blood immune cells (Fig. S1b).

Behavioural testing

Behavioural testing was performed on one female and one male offspring per litter, at 12 weeks old. The tests conducted were open field and sociability as previously described (Loring et al. 2021), performed consistently in that order and between 11:00 and 14:00 hours each assessment day. Briefly, open field test is used to quantify locomotor activity and anxiety-like behaviour under stress-inducing conditions. For this, the mouse was placed in the southwest corner of a 40 cm × 40 cm plexiglass box with overhead light, for 5 min and movement into the outermost (darker) zone of the box is monitored (see Fig. S3a). Specifically, number of entries into, time spent within, and travel distance and speed in the demarked outer zone are recorded. Sociability test is employed to investigate social and autistic-like behaviours. For this, the mouse is placed in the centre of a rectangular plexiglass box consisting of three-chambers (each 20 cm × 40 cm), connected by up-sliding doors which are opened right before the start of recording. The sociability test consists of 3–5 min observations referred to as habituation, sociability and preference for social novelty (see Fig. S3b). At the end of each observation, the tested mouse is herded to the centre chamber and the door is closed. For habituation, only the test mouse was present in the maze. To measure sociability, a stranger mouse (age- and sex-matched) in a wire cage was placed in the left chamber. To measure preference for social novelty, two mice were placed at the left (familiar) and right chamber (stranger). The number and duration of the interactions were recorded manually when the tested mouse sniffed, interacted with, or climbed on the occupied cages. All testing was recorded and analysed using ANY-maze (Stoelting Co., Wood Dale, IL, USA).

Statistical analysis

Data are represented as mean ± s.e. of mean (s.e.m.) or mean ± s.d. as indicated. Statistical analysis was performed by either GraphPad Prism 9 (GraphPad Software LLC, San Diego, CA, USA), or IBM SPSS Statistics for Windows ver. 28.0.0.0 (IBM Corp, Armonk, NY, USA). The data was checked for Gaussian distribution (normality) and homoscedasticity (equal variance), to confirm that the assumptions of a linear model were met. The effect of age on litter size throughout female reproductive lifespan was analysed using a repeated measure mixed model analysis, followed by post hoc pairwise comparisons to determine differences in litter size between two consecutive months. The effect of age on litter size, pregnancy rate, livebirth rate, newborn death, pup survival, and maternal immune cell distribution were analysed by unpaired Student’s t-test or Fisher’s Exact test as indicated. Pup sex ratio was analysed using a two-tailed binomial test to compare each group’s observed sex ratio to the expected distribution of 0.5 for both sexes. Offspring postnatal growth trajectory, glucose metabolism, organ weight and immune cell phenotype were calculated by a multilevel, repeated measures mixed model analysis, with post hoc Fisher’s l.s.d. To understand possible predictors of the dependent variables studied, some analysis used either litter size (postnatal weight gain and behavioural testing) or body weight (glucose metabolism, organ weight and peripheral blood immune cell composition) as covariates at the time of the analysis. Litter (mother ID) was also introduced into the model to check for random effects over the covariance structure of the data, as per Golub and Sobin (2020).

Ethical statement and mouse husbandry

All procedures were approved by the University of Adelaide Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Animals were maintained at 24°C on a 12-h light, 12-h dark cycle, with Envigo standard rodent diet (Teklad Global Rodent Diet® #0915: 19% (w/w) crude protein, 9% (w/w) fat, 45% (w/w) carbohydrate; Madison, WI, USA) and water available ad libitum.

Parental reproductive aging is characterised by smaller litters and higher newborn death

Reproductive performance in mice within a breeding colony was monitored and showed a significant effect of the age of the parents on litter size. Month to month pairwise comparisons revealed that the first significant decrease in litter size occurred when the mice reached 11 months old, presenting an average litter size of 4.7 ± 2.3 pups that was lower than the average of 5.5 ± 3.0 pups at 10 months old (Fig. 1a). Thus, we used this information to classify mice as either young (< 11 months old) or old (≥ 11 months old). When offspring survival to the age of weaning (21 days old) was analysed using this classification, it was found that pup survival was reduced when the dams were older (Fig. 1b). Pup mortality rate was almost doubled when the mice were older compared to when they were young (27.5% vs 14.4% respectively; Fig. 1b). These data indicate that the effects of aging on mouse breeder pairs’ reproductive success are first seen at 11 months old and can be identified by smaller litters, as well as reduced pup survival.

Fig. 1.

Fecundity declines with parental age. (a) Litter size (i.e. number of pups born) over the lifespan of continuously housed male and female (1:1) breeder pairs. Black data points indicate ages at which mice were considered ‘young’, while grey data points indicate the ages after which there was a first significant reduction in litter size compared to the previous month, and mice were then considered ‘old’ (N = 48 female breeders; *P < 0.05 compared to 10-month-old time point). (b) Pup survival to weaning at 21 days post-birth in litters from female breeders at < 11 months old (black bar) compared to those born when females were ≥ 11 months old (grey bar) (N = 2063 pups from < 11 months, 592 pups from ≥ 11 months; ****P < 0.0001). Data was analysed using repeated measure mixed model with post hoc paired t-test comparisons test (a) or Fisher’s Exact test (b).

Aging impairs female fecundity and offspring survival

We next assessed the effects of reproductive aging on fecundity, pup survival and offspring phenotypes using C57BL6/J female mice. Reproductively old female mice (12 month old and multiparous) and young females (3 months old and nulliparous) were each paired (1:1) with 8–12 weeks old stud males to assess pregnancy and birth rates (Fig. 2a). Older females became pregnant at a rate that was not statistically different to that of young females; however, only 34.3% of pregnant old females gave birth to live pups, compared to 100% of pregnant young females (Table 1). The older females also had smaller litters, averaging 2.6 ± 1.3 pups per litter, compared to 6.9 ± 2.3 pups per litter in younger mice (Table 1). In addition, fewer of the offspring from older females survived to weaning, compared to young females (79.3% vs 96.9%, respectively; Table 1). Interestingly, offspring from young females were born at a sex ratio which was female-biased; while litters from older females had balanced sex ratios that were not different from the expected 50:50. It is not clear whether these sex ratio differences were present in newborns or due to sex-specific differences in pup death. These results show that early reproductive decline impairs female reproductive success and offspring survival.

Fig. 2.

Female offspring from older mothers are heavier. (a) Young (3 months) and reproductively aged (12 months) female mice were paired with young male mice to produce offspring. Offspring were assessed for body weight up to 16 weeks of age, and underwent behavioural, glucose and insulin tolerance, and immune cell phenotyping at the indicated ages. Body weight over time (from 3 to 16 weeks of age) for female (b) and male offspring (c) born to young females (black line) or older females (grey line). N = 77 pups from 11 young, 27 pups from 6 old dams/litters. Data are presented as mean ± s.e.m. Statistical analysis was a multilevel, repeated measures mixed model using litter size at birth as a covariate with post hoc Fisher’s Least Significant Difference test. *P < 0.05, **P < 0.01, ***P < 0.001.

Maternal age at conception alters growth trajectory in female offspring

Postnatal growth of offspring was examined by measuring body weight from 3 to 16 weeks of age (Fig. 2a). Female offspring of old mothers were on average 2.3 g heavier than those born to young mothers, a difference that was statistically significant even accounting for the differences in litter size as a co-variate (Fig. 2b). In contrast, male offspring from older females had a growth trajectory that was not different from those born to young females (Fig. 2c). At 16 weeks of age, final body weight was recorded, and key organs collected and weighed, namely reproductive tract (uterus plus ovaries in females), testis (males), abdominal fat pad, spleen, liver, kidneys and heart. Female offspring born to older mothers were on average 3.54 g heavier than those from young, presenting 52% heavier abdominal fat pads (0.79 g vs 0.38 g respectively), and 19% lighter kidneys (0.32 g vs 0.26 g respectively) than those from young (Table S3). However, these relative differences in organ weights disappeared when adjusting for body weight as a co-variate, since the female offspring from older mothers are heavier overall (Table S3). Male offspring from both groups had similar body weights at 16 weeks of age; however, when organ weight was adjusted to body weight as a co-variate, males born to old mothers had 17% lighter abdominal fat pads and 5% heavier kidneys than those from young (Table S3).

At the same time, blood was collected for analysis of immune cell distribution in serum (see gating strategy in Fig. S1a). The older dams had lower CD4⁺ T cell and higher neutrophil proportions in their peripheral blood immune cell populations (Table S4), as expected (Levenson et al. 2020). There were no differences in the proportions of specific immune cell types in the male offspring from old mothers compared to young mothers (Table 2). However, female offspring of older mothers presented 9.3% higher proportion of circulating B cells (Table 2). Together, these findings indicate that the age of the mother at conception has a sex-distinct influence on offspring growth, but only a modest effect on adult offspring immune cell distribution.

Maternal age alters glucose tolerance in male offspring

The offspring underwent metabolic assessments, namely glucose tolerance tests (GTT) at 14 weeks of age and insulin tolerance tests (ITT) at 15 weeks old (Fig. 2a). The age of the mother had no effect on female offspring glucose tolerance or insulin sensitivity (Figs 3a, b and S2a, b). Differences were apparent, however, in male offspring. Circulating glucose levels in male offspring from older mothers were on average 2.63 mmol/mL lower at 15, 30 and 60 min post-glucose challenge (Fig. 3c). This was confirmed by a lower area under the curve result for male offspring of older females (Fig. S2c). During the insulin tolerance test, the male offspring of older mothers exhibited an average 1.5 mmol/mL lower baseline (fasting) levels (0 min) compared to those from young (Fig. 3d). However, the relative response to insulin and the area under the curve result were not different between the two groups (Fig. 3d and S2d). These results suggest that maternal aging influences glucose tolerance in the male offspring.

Fig. 3.

Maternal aging alters glucose tolerance in male offspring. Blood glucose level at 0, 15, 30, 60 and 120 min post-glucose challenge in female (a) and male (c) offspring at 15 weeks old. Blood glucose level at 0, 15, 30, 60 and 120 min post-insulin challenge in female (b) and male offspring (d) at 15 weeks old. N = 77 pups from 11 young, 27 pups from 6 old dams/litters. Data are presented as mean ± s.e.m. Statistical analysis was multilevel, repeated measures mixed model, using body weight as a covariate with post hoc Fisher’s Least Significant Difference test. **P < 0.01, ***P < 0.001.

Offspring from older mothers display hyperactivity

At 12 weeks of age, the offspring underwent behavioural testing to assess neurodevelopment for anxiety-like and autistic-like behaviour (Fig. 2a). In open field tests, mice with anxiety-like phenotypes spend most of the time in the outer (darker) zone (see Fig. S3a). Female offspring from both groups displayed similar behaviours during the open field assessment (Fig. 4a–d). In contrast, male offspring born to old females travelled farther (14.87 cm vs 9.68 cm; Fig. 4g) and faster (0.056 m/s vs 0.036 m/s; Fig. 4h) in the outer zone compared to those born to young mothers. The offspring from older versus younger mothers did not exhibit any differences in behaviour during the sociability tests (Fig. S3b–n). Taken together, these results indicate that male offspring from older mothers showed hyperactivity, a marker of anxiety-like behaviour.

Fig. 4.

Maternal aging increases anxiety markers in male offspring. Open field test assessments in female (a–d) and male (e–h) offspring at 12 weeks old. Number of entries to- (a, e), time spent in- (b, f), distance travelled in- (c, g) and average speed in- outer zone (d, h) recorded. N = 13 pups from seven young, eight pups from 4 old dams/litters (one female and one male offspring per litter). Data are represented as mean ± s.e.m. Statistical analysis was a multilevel mixed model, using litter size as a covariate with post hoc Fisher’s Least Significant Difference test. *P < 0.05; **P < 0.01.