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Spermidine Rejuvenates Oocyte Quality by Enhancing Mitophagy During Female Reproductive Aging

Aging is a key risk factor for infertility in individuals with ovaries due to a decrease in ovarian reserve and egg quality. The significance of metabolic processes in reproductive aging remains unclear. Our study used untargeted metabolomics and identified spermidine as an essential metabolite that protects eggs from aging. We observed lower levels of spermidine in the ovaries of older mice. We found that supplementing with spermidine improved these mice’s follicle development, egg maturation, early embryo development, and fertility.

Additionally, spermidine was shown to enhance the quality of eggs by improving mitophagy and mitochondrial function, a finding echoed in pig eggs under oxidative stress. These results indicate that spermidine supplementation could improve egg quality and fertility outcomes for those trying to conceive later in life. Further research is necessary to determine the safety and effectiveness of this treatment in humans.

Human fertility and reproductive potential decline significantly with age, particularly after the mid-30s. By age 34, about 10% of individuals seeking to conceive naturally may face infertility, which soars to 87% by age 45. Older prospective parents are more likely to encounter infertility, miscarriages, perinatal deaths, and congenital disabilities. While assisted reproductive technologies (ART) offer some solutions, their success rates also diminish with age. Women under 35 years see a 41-43% chance of live births through in vitro fertilization (IVF), but this plummets to 1-2% for those 44 and older due mainly to declining ovarian reserve, egg quality, and increased chromosomal abnormalities. Recent research indicates that nicotinamide mononucleotide (NMN) supplementation may improve egg quality and combat fertility decline in older female mice. Still, more effective compounds and methods are yet to be identified.

About Spermidine Molecule

Polyamines, including putrescine, spermidine, and spermine, are ubiquitously occurring polycations that are synthesized in almost every living cell and exist in many tissues, organs, and organisms. They bind and stabilize negatively charged molecules such as DNA, RNA, proteins, and adenosine triphosphate (ATP) to participate in diverse physiological and pathophysiological processes. Of these, spermidine is an intermediate polyamine compound that is synthesized from putrescine via the action of decarboxylated S-adenosyl methionine through spermidine synthase (SRM) and acts as a precursor of spermine by the action of spermine synthase (SMS). Spermidine was initially isolated from semen and is also found in most cells and tissues, including the ovary15,16. It participates in a wide range of cellular events, including regulation of transcription and translation, induction of oxidative stress, autophagy and apoptosis, and maintenance of genomic stability because of its anti-inflammatory activities, antioxidative properties, reinforced mitochondrial functions, and enhanced proteostasis. It has been reported that spermidine administration prolongs lifespan in yeast, flies, worms, and human immune cells by induction of autophagy18,19. In addition, dietary supplementation with spermidine suppressed age-induced memory impairment in Drosophila20, prevented neurodegenerative diseases with TAR DNA-binding protein 43 (TDP-43) proteinopathies in mice, and exerted cardioprotective effects by reducing cardiac hypertrophy and decline in diastolic function in old mice22. Although many studies have been reported about the vital role of spermidine for aging in somatic cells, the effect of spermidine on oocyte aging has not been clarified.

In the current study, we took advantage of untargeted metabolome analysis to discover that spermidine levels were reduced in the ovaries of aged mice, and we found that supplementation with spermidine in vivo rejuvenated the quality of oocytes from old mice by promoting their maturational competency, fertilization potential, and embryonic development ability, thereby increasing animal fertility. Additionally, we determined that spermidine recovered mitophagy activity to inhibit apoptosis during oocyte aging by micro transcriptome analysis.

Oocyte Quality Results

The metabolome reveals reduced spermidine levels in ovaries of aged mice

We compared ovarian metabolome profiles between young and aged mice to investigate how advanced age impacts metabolite changes in ovaries (Supplementary Data 1). Principal-component analysis revealed that five biological replicates clustered together in each group but were separated far from each other between young and aged samples (Fig. 1a). Heatmap analysis of differential metabolites showed that ovarian metabolome profiles were substantially altered in aged mice compared to those in young ones (Fig. 1b). In addition, a volcano plot displays the number of upregulated and downregulated metabolites in the aged group compared to the young counterpart (Fig. 1c). We further ranked the top ten pathways that were substantially altered in ovaries from aged mice compared to those of young ones by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Fig. 1d). Among them, one pathway was upregulated and nine pathways were down regulated as analyzed by differential abundance score (Fig. 1e). Notably, we found that spermidine, an intermediate polyamine compound that takes part in a variety of cellular processes, was present in both β-alanine metabolism and ATP-binding cassette (ABC) transporter pathways as a downregulated metabolite induced by aging (Fig. 1f, g). Also, the bar graph obtained from metabolome data showed that the level of spermidine was significantly reduced in the ovaries of aged mice (Fig. 1h). Collectively, our metabolome data uncover the fact that spermidine is a critical metabolite that declines in the ovaries of aged mice.

Metabolome profiling of ovaries in aged mice

Fig. 1 | Metabolome profiling of ovaries in aged mice. a, Principal-component(PC) analysis of samples from ovaries of young and aged mice. Blue circles represent samples from the young group, and red circles represent samples from the aged group. b, Heatmap illustration shows differential metabolites in ovaries from young versus aged mice. c, Volcano plot displays differential metabolites (upregulated, red; downregulated, blue) in ovaries from aged mice compared to those from young ones. d, KEGG enrichment analysis of differential
metabolites in ovaries from aged mice compared to those from young controls. CoA, coenzyme A. e, Differential abundance (DA) score analysis of differential metabolites (upregulated, red; downregulated, blue) in ovaries from aged mice compared to those from young ones. f, Heatmap illustration displays differential metabolites in the β-alanine metabolism pathway in ovaries from young and
aged mice. g, Heatmap illustration displays differential metabolites in the ABC transporter pathway in ovaries from young and aged mice. h, Metabolome data show levels of spermidine in ovaries from young and aged mice. ***P = 0.0005. Data are expressed as mean percentage (mean ± s.e.m.) of five biologically independent samples. Statistical significance was determined by two-tailed
unpaired t-test.

Spermidine improves ovarian development and female fertility of aged mice

To further validate whether aging would reduce spermidine levels in ovaries, we performed an enzyme-linked immunosorbent assay-based spermidine-detection assay using mouse ovarian lysates. The enzyme-linked immunosorbent assay results showed that a substantial decrease in spermidine was observed in ovaries from aged mice compared to those from young controls (Fig. 2b). Strikingly, supplementation with spermidine by intraperitoneal injection remarkably increased the spermidine level in ovaries from aged mice. It promoted the maturational rate of oocytes (Fig. 2a,b and Extended Data Fig. 1a), highlighting the potential role of spermidine in improving oocyte quality and female reproductive lifespan in animals of advanced age. We next evaluated ovarian development following spermidine supplementation in aged mice by hematoxylin and eosin staining of ovarian sections. We found that follicles at different developmental stages displayed normal morphology in ovaries from young mice (Fig. 2c and Extended Data Fig. 1b). On the contrary, a large number of degenerated follicles were present in ovaries of aged mice but were partially recovered in ovaries of spermidine-supplemented mice as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Fig. 2e,f). Consistently, quantification of follicle number at different developmental stages revealed that spermidine supplementation increased the number of pre-antral and antral follicles in ovaries of aged mice (Fig. 2d). Consequently, the fertility test showed that the litter size of female mice was considerably lowered by aging, whereas it was restored to some degree after spermidine supplementation (Fig. 2g,h). Altogether, these data suggest that replenishment of spermidine is a feasible strategy to improve oocyte and ovarian development and female fecundity of aged animals.

Spermidine enhances the in vivo maturation competency of oocytes from aged mice

As the development of ovaries from aged mice was restored by supplementation with spermidine, we then assessed its effects on oocyte quality. After superovulation, we collected oocytes for counting and evaluating morphology. We found that the number of ovulated oocytes and their maturational rate as judged by extrusion of the first polar body (PB1) from aged mice was dramatically lower than that in the young ones, but the incidence of fragmentation was significantly increased (Fig. 3a–d). By contrast, supplementation with spermidine rescued the defects in the quantity and quality of oocytes from aged mice (Fig. 3a–d).

Given that aneuploidy is one of the key hallmarks of aging-induced low-quality oocytes, spindle–chromosome structure and chromosome number were further examined. By immunofluorescent staining and confocal imaging, we observed that barrel-like spindle apparatuses with well-aligned chromosomes were present in most oocytes from young mice, while various morphology-aberrant spindles with misaligned chromosomes were found in oocytes from aged mice (Fig. 3e). Notably, some spindle–chromosome abnormalities in oocytes from aged mice were restored following spermidine supplementation (Fig. 3e). Quantification data also showed that the proportions of abnormal spindles and misaligned chromosomes were considerably higher in oocytes from aged mice than those from young ones but were reduced in oocytes from spermidine-supplemented mice (Fig. 3f,g). In line with these observations, the results of chromosome spreading and counting showed that the frequency of aneuploidy, as assessed by more than or less than 20 univalents, remarkably grew in oocytes from aged mice in comparison with that of young controls, whereas it declined with spermidine supplementation (Fig. 3h,i). Our observations indicate that spermidine could ameliorate aging-induced meiotic defects during oocyte nuclear maturation.

Spermidine recovers the fertilization capacity and early embryonic development potential of oocytes from aged mice

Aside from nuclear maturation, cytoplasmic maturation is another pivotal process that determines the quality of oocytes and is highly related to subsequent fertilization and embryonic development.

We thus investigated the dynamics of cortical granules (CGs) and mitochondria, two critical indicators of oocyte cytoplasmic maturation. By fluorescent imaging and intensity measurement, we found that the signals of CGs on the subcortical region of oocytes as shown by lens culinaris agglutinin, fluorescein (LCA–FITC) staining were prominently reduced in oocytes from aged mice compared to those in young ones and were effectively restored in the spermidine-supplemented group (Fig. 4a,b). Consistent with this observation, spermidine supplementation also maintained the level of lovastatin, a key component of CGs associated with sperm-binding ability, in the subcortex of oocytes from aged mice (Extended Data Fig. 2a,b). Furthermore, staining of mitochondria with Mito Tracker Red revealed that mitochondria were distributed evenly in the cytoplasm with accumulation around the spindle in oocytes from young mice (Fig. 4c,d and Extended Data Fig. 2c). However, aging compromised this distribution pattern of mitochondria, showing faded signals in the cytoplasm and loss of aggregation around chromosomes, which was rescued by spermidine supplementation to some extent (Fig. 4c,d).

Because the anomalous dynamics of CGs and ovastacin may impair the sperm-binding ability of oocytes, we next tested this in the sperm–oocyte binding assay. Counting of sperm head binding to the zona pellucida (ZP) of oocytes indicated that oocytes from young mice supported robust binding of an abundance of sperm before fertilization but lost it after fertilization (Fig. 4e,f). By striking contrast, the number of sperm binding to oocytes from aged mice was remarkably lower even before fertilization, indicative of the weakened sperm-binding ability caused by aging (Fig. 4e,f). Similarly, spermidine supplementation increased the number of sperm binding to oocytes from aged mice. In the meantime, we also demonstrated that spermidine supplementation restored rates of in vitro fertilization and blastocyst formation that were markedly decreased in oocytes from aged mice (Fig. 4g–i). Therefore, our results imply that spermidine strengthens the fertilization capacity and embryonic development potential of oocytes from aged mice by promoting their cytoplasmic maturation.

To further verity the beneficial action of spermidine on oocytes from aged mice, dietary supplementation with spermidine in drinking water was performed. We found that the quality of oocytes from aged mice including maturation competency, spindle–chromosome structure, chromosome euploidy, fertilization capacity and early embryonic development potential was prominently improved by dietary supplementation with spermidine (Extended Data Fig. 3), demonstrating that spermidine supplementation by different administration methods enhances the quality of oocytes impaired by aging.

Spermidine boosts in vitro maturation of oocytes from aged mice

Because supplementation with spermidine could enhance in vivo maturation of oocytes from aged mice, we further asked whether it had a favorable effect on in vitro maturation by supplementing spermidine at different concentrations in the culture medium to observe PB1 extrusion in oocytes. Statistical data indicated that supplementation with 50 μM spermidine significantly recovered PB1 extrusion after in vitro maturation of germinal vesicle (GV) oocytes from aged mice (Fig. 5a,b and Extended Data Fig. 4a). In addition, in most oocytes from young

mice, the spindle apparatus normally assembled around well-aligned chromosomes at metaphase I, with the correct attachment of kinetochores on chromosomes by microtubule fibers (Fig. 5c–g). While a higher percentage of disorganized spindles, misaligned chromosomes and improper attachment of kinetochores and microtubules was present in oocytes from aged mice, which was considerably decreased by spermidine supplementation (Fig. 5c–g). Accordingly, spindle–chromosome structure at metaphase II after in vitro maturation was also disrupted by aging and restored following spermidine supplementation

(Extended Data Fig. 4b–d). As a result, spermidine supplementation reduced the occurrence of aneuploidy from ~40% to ~20% in oocytes at metaphase II from aged mice (Fig. 5h,i). In conclusion, we demonstrate that supplementation with spermidine during in vitro maturation also rejuvenated the quality of oocytes from aged mice.

Polyamine metabolite spermidine rejuvenates oocyte quality by enhancing mitophagy during female reproductive aging

Microtranscriptomic analysis reveals regulatory pathways involved in spermidine effects on oocytes from aged mice

To gain insight into potential mechanisms regarding how spermidine improves the quality of oocytes from aged mice, micro transcriptomics was carried out to compare changes in transcript levels (Supplementary Data 2). Heatmap analysis showed remarkably different

transcriptomic profiling between oocytes from young and aged mice, and the profile was partially restored in oocytes from spermidine supplemented mice (Fig. 6a). Volcano plots and fold-change graphs further revealed that 516 upregulated and 307 downregulated transcripts were present in oocytes from aged mice compared to those from young ones (Fig. 6b and Extended Data Fig. 5a); furthermore, 122 upregulated and 136 downregulated transcripts were observed in oocytes from spermidine-supplemented mice compared to those from aged ones (Fig. 6c and Extended Data Fig. 5b). Additionally, a Venn diagram presents the overlapping differentially expressed genes (DEGs) between these two groups (Extended Data Fig. 5c). There were also some DEGs between oocytes from spermidine-supplemented mice compared to those from young ones (Extended Data Fig. 5d). By performing KEGG analysis, we noticed that transcripts of DEGs were enriched in pathways related to oxidative phosphorylation and autophagy in oocytes from aged mice in comparison with those from young counterparts, which was rescued by spermidine supplementa￾tion (Fig. 6d,e). KEGG network analysis also indicated that these DEGs in the autophagy and mitophagy pathways might be associated with the apoptosis pathway (Extended Data Fig. 5e,f). In addition, gene ontology (GO) analyses in both oocytes from aged mice compared to young controls and oocytes from spermidine-supplemented mice compared to those from aged ones showed that DEGs were enriched in biological processes such as apoptosis, cell cycle and mitochondrial organization (Fig. 6f,g). Therefore, from the above observations, we hypothesize that the improvement in oocyte quality from aged mice induced by spermidine supplementation might be mediated through autophagy and mitochondrial functions.

Spermidine restores autophagy in oocytes from aged mice

A heatmap of DEGs enriched in the autophagy pathway indicated that spermidine supplementation to some degree recovered transcript levels of 26 genes in oocytes from aged mice (Fig. 7a). We randomly selected four of them to verify their mRNA levels in each group of oocytes by PCR with reverse transcription (RT–PCR), and the results were consistent with the RNA-seq data (Fig. 7b,c). As autophagy is featured by the formation of autophagosomes that fuse with lysosomes to form autolysosomes, we next examined autophagy by staining for the autophagosome marker microtubule-associated protein 1A or 1B light chain 3 (LC3). Fluorescence imaging and quantification results illustrated that a great number of LC3 signals with many large foci were present in oocytes from young mice (Fig. 7d,e). By striking contrast, aging reduced both amount and size of LC3 foci, and this was recovered by spermidine supplementation (Fig. 7d,e). Furthermore, we validated autolysosome formation by double staining with antiLC3 antibody and LysoTracker Green. A colocalization pattern of LC3 foci and lysosome vesicles was observed in oocytes from young and spermidine-supplemented mice but was lost in those from aged ones (Fig. 7f). Quantitatively, both Pearson’s correlation coefficient (PCC) and Mander’s correlationcoefficient (MCC) analyses showed that the relationship between signals from LC3 and lysosomes became much weaker in oocytes from aged mice compared to that from young ones, and it was strengthened by spermidine supplementation (Fig. 7g and Extended Data Fig. 6a,b). Thus, our data indicate that spermidine is able to elevate autophagy in oocytes from aged mice.

Spermidine enhances mitophagy and mitochondrial function in oocytes from aged mice

Mitophagy is a specialized form of autophagy that mediates the removal of dysfunctional mitochondria to maintain both mitochondrial quantity and quality. Taking advantage of our RNA-seq data, we displayed mRNA levels of 11 DEGs related to the mitophagy pathway in a heatmap, indicating that spermidine supplementation rescued the change in transcript levels of these DEGs in oocytes from aged mice (Fig. 8a). Also, RT–PCR results of two selected DEGs further confirmed the transcriptomic data (Fig. 8b, c). We then tested the formation of mitophagosomes by double staining for voltage-dependent anion channel 1 (VDAC1), a pivotal protein required for PTEN-induced kinase 1 (PINK1)–parkin-mediated mitophagy, and LC3. PCC and MCC analyses based on the fluorescence signals demonstrated that most VDAC1 signals colocalized with LC3 foci in the cytoplasm of oocytes from young mice, but only a few VDAC1 signals colocalized with LC3 foci in oocytes from aged mice (Fig. 8d,e and Extended Data Fig. 6c,d). On the contrary, spermidine dramatically promoted colocalization of VDAC1 and LC3 signals in oocytes from aged mice (Fig. 8d,e and Extended Data Fig. 6c,d), implying that the impaired mitophagosomes caused by aging in oocytes can be restored after spermidine supplementation. In addition, the formation of mitolysosomes in each group of oocytes was assessed by double staining with MitoTracker Red and LysoTracker Green. Similarly, colocalization of mitochondria and lysosomes was compromised by aging in oocytes and could be recovered by supplementation with spermidine as evaluated by PCC and MCC analyses of fluorescence signals (Fig. 8f,g and Extended Data Fig. 6e,f). These findings illustrate that spermidine reinforces mitophagy activity in oocytes from aged mice.

To investigate whether mitophagy enhancement would strengthen mitochondrial function, we detected mitochondrial membrane potential (ΔΨm) by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzi midazolylcarbocyanine iodide ( JC-1) staining. The ratio of J-aggregates at high ΔΨm to JC-1 monomers at low ΔΨm was found to be high in oocytes from young mice but decreased in those from aged mice (Fig. 8h,i). By comparison, spermidine supplementation substantially elevated the ΔΨm level in oocytes from aged mice (Fig. 8h,i), indicative of improvement in mitochondrial function. In line with previous studies by us and others9,23,24, we observed that mitochondrial dysfunction in oocytes caused by aging produced excessive reactive oxygen species (ROS), accumulated DNA damage and high incidence of apoptosis as evaluated by fluorescence imaging and intensity quantification (Extended Data Fig. 7). As expected, spermidine supplementation effectively reduced the levels of ROS and DNA damage, thus inhibiting apoptosis (Extended Data Fig. 7). In sum, we demonstrate that spermidine strengthens mitochondrial function through activation of mitophagy.

Inhibition of mitophagy activity interferes with the spermidine-enhanced quality of oocytes from aged mice

To further validate that the favorable effects of spermidine on oocytes from aged mice were mediated by mitophagy, we cultured oocytes from aged mice in a maturation medium containing both spermidine and the mitophagy inhibitor Mdivi-1 to assess oocyte maturation. The concentration of Mdivi-1 was determined by observing the rate of PB1 extrusion and the intensity of LC3 signals in oocytes from young mice (Extended Data Fig. 8a–c). Notably, in the presence of Mdivi-1, the maturation rate of oocytes from aged mice treated with spermidine as evaluated by PB1 extrusion was even lower than that of aged ones (Extended Data Fig. 8d). Furthermore, measurement of fluorescence intensity of LC3 signals indicated that Mdivi-1 treatment also impaired spermidine-induced mitophagy recovery in oocytes from aged mice (Extended Data Fig. 8e,f), suggesting that mitophagy activity is required for spermidine effects on oocytes from aged mice.

Improvement of spermidine-induced oocyte quality is conserved across species

We next used porcine oocytes as a model to test the potential effects of spermidine on oocyte maturation in another species. As shown in Extended Data Fig. 9a,b, in vitro maturation of porcine oocytes as assessed by PB1 extrusion was substantially lower in the H2O2-treated group than in the controls, and maturation was restored after spermidine supplementation (Extended Data Fig. 9a,b). Furthermore, we found that spermidine supplementation recovered LC3 levels and mitochondrial dynamics in H2O2-treated porcine oocytes (Extended Data Fig. 9c–f), thus removing excessive ROS and inhibiting apoptosis (Extended Data Fig. 9g–j). Therefore, we demonstrate that the mechanism of action of spermidine on oocyte development under oxidative stress is highly conserved.


Ovarian development in mammals is an orderly, coordinated, complicated and finely regulated physiological process that has been affected by multiple intrinsic and extrinsic factors to produce high-quality oocytes and thus guarantee female reproduction25. Among various contributors, metabolic regulation is one of the most pivotal factors. It has been widely reported that metabolic disorders can result in ovarian dysfunctions and affect female fertility. For instance, polycystic ovarian syndrome is a common metabolic disorder in reproductive-age persons. In addition, reproductive aging may impact female fertility by impairing metabolic homeostasis28. However, our current understanding regarding how metabolic changes influence the deterioration of germ cells during reproductive aging remains largely elusive.

To this end, we carried out untargeted metabolomics to compare metabolic profiles in ovaries between young and aged mice. Our metabolomic data identified that spermidine was remarkably reduced in the ovaries from aged mice. Spermidine has been found to function as an autophagy inducer to participate in a diversity of cellular processes associated with aging progression across species. Therefore, we proposed that spermidine might be a key metabolite in maintaining the female reproductive lifespan. Interestingly, we also noticed that levels of several steroids such as pregnenolone, progesterone, and medroxyprogesterone, were dramatically upregulated in ovaries of aged mice. However, a previous study has shown

that plasma concentrations of these three steroids as measured by LC–MS/MS decline with advanced age in humans30. Whether this discrepancy is attributed to different tissue sources or species requires further investigation. In addition, there is conflicting evidence regarding the effects of progesterone levels on oocyte quality. It has been reported that the number of oocytes obtained during intracytoplasmic sperm-injection procedures is positively correlated with serum progesterone levels but negatively associated with follicular fluid progesterone levels. Moreover, high progesterone levels in human follicular fluid are positively correlated with oocyte quality as defined by subsequent embryonic development, implantation and pregnancy but paradoxically are associated with polyspermy and multipronuclear embryos. It appears that both reduced and excessive progesterone levels are detrimental to oocyte quality.

Female aging has been reported to compromise both nuclear and cytoplasmic maturation of oocytes, including polar body extrusion, chromosome euploidy, CG dynamics, mitochondrial function, fertilization capacity, and early embryonic development potential. However, the degrees of influence fluctuate in different studies. As our data presented similar defects induced by aging in mouse oocytes, we then supplemented aged mice with exogenous spermidine to assess its potential effects on oocyte quality, ovarian function, and female fertility. Our findings validated that in vivo supplementation with spermidine promoted oocyte maturation, fertilization and embryonic development, thereby increasing the fertility of aged mice. In addition, spermidine supplementation in the culture medium also enhanced the quality of oocytes from aged mice during in vitro maturation, documenting that spermidine can rejuvenate oocyte quality both in vivo and in vitro. It is worth noting that excessive spermidine is not necessarily beneficial to female reproduction, as shown by our data that supplementation with 100 mg per kg spermidine resulted in a slower oocyte-maturation rate than that with 50 mg per kg spermidine. This is consistent with a previous study that found that supplementation with spermidine at supraphysiological doses in young female mice causes ovarian oxidative stress and induces granulosa cell apoptosis. A similar phenomenon was observed in our recent study in that supplementation with NMN at higher doses did not obtain an optimal outcome for oocyte-maturation competency in aged mice. As for the comparison between spermidine and NMN with regard to female reproductive improvements, it could not be made based on the current data, as we used mouse models at different ages in these two studies. The comparison or combination effects of spermidine and NMN with regard to oocyte quality and animal fertility need to be clarified in future work.

To gain insights into the mechanisms underlying spermidine’s effect on oocytes from aged mice, we performed microtranscriptomic analysis to characterize potential downstream effectors. In agreement with previous studies that spermidine induces autophagy to protect against age-related pathologies18,40, our RNA-seq data indicated that autophagy, mitophagy and mitochondrial function were the main pathways by which spermidine restored oocyte quality in aged mice. Subsequent investigations verified that spermidine elevated autophagy to restore mitophagy activity and mitochondrial function in oocytes from aged mice, hence inhibiting excessive ROS production and apoptosis induced by aging.

As one of the important hallmarks of oocyte aging is the generation of oxidative stress, we then used H2O2 treatment to partially mimic the phenotypes induced by aging in porcine oocytes, including oocyte meiotic failure, mitochondrial dysfunction, impaired autophagy and apoptosis, certifying that the beneficial effects of spermidine on oocyte maturation were conserved across species. Intriguingly, spermidine has been identified as an important mating component to promote fertilization in an autophagy-independent manner in Saccharomyces cerevisiae and Caenorhabditis elegans41, suggesting that spermidine might regulate reproductive processes through multiple mechanisms.

In sum, based on ovarian metabolomic data and much in vivo and in vitro experimental evidence, we demonstrate that spermidine is a critical metabolite for preserving oocyte quality during aging. Supplementation with spermidine can promote oocyte maturation, enhance

fertilization capacity as well as early embryonic development potential and thus increase female fertility in aged animals. Notably, spermidine strengthens mitophagy activity to maintain mitochondrial function and to suppress apoptosis in oocytes from aged mice (Supplementary Fig. 1). Thus, our study provides clinical implications for the potential application of spermidine to ameliorate the reproductive outcome of women at an advanced age by means of either natural pregnancy or assisted reproductive technology.


The detailed methods are downloadable in this pdf file.  It includes the following topics:

  • Mice and spermidine supplementation
  • Metabolomic analysis
  • Antibodies and dyes
  • Measurement of spermidine levels
  • Oocyte collection and maturation
  • Histological analysis and follicle quantification
  • Fluorescence staining and measurement
  • Chromosome spreading and kinetochore staining
  • Sperm-binding assay
  • In vivo fertilization and in vitro embryo culture
  • Microtranscriptome sequencing and RNA library construction
  • RNA isolation and quantitative real-time PCR
  • Porcine oocyte collection and in vitro maturation
  • Statistics and reproducibility

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