Preservation of ovarian function using human pluripotent stem cell-derived mesenchymal progenitor cells.

Ovarian reserve diminishes with age, and older women experience a corresponding shift in sex hormone levels. These changes contribute to an age-dependent decrease in fertility and a decline in overall health. Furthermore, while survival rates following cancer treatment have improved for young female patients, a reduction in ovarian function due to the side effects of such treatments can be difficult to avoid. To date, no effective therapy has been recommended to preserve ovarian health in these patients. Mesenchymal progenitor cells (MPCs) are considered a promising option for cell therapy aimed at maintaining fertility and fecundity. Although MPCs derived from human adult tissues are recognized for their various protective effects against ovarian senescence, they are limited in quantity. Consequently, human pluripotent stem cell-derived MPCs (hPSC-MPCs), which exhibit high proliferative capacity and retain genetic stability during growth, have been utilized to delay reproductive aging. This review highlights the impact of hPSC-MPCs on preserving the functionality of damaged ovaries in female mouse models subjected to chemotherapy and natural aging. It also proposes their potential as a valuable cell source for fertility preservation in women with a variety of diseases.

Multiple treatments with human embryonic stem cell-derived mesenchymal progenitor cells preserved the fertility and ovarian function of perimenopausal mice undergoing natural aging.

OBJECTIVES: Currently, no approved stem cell-based therapies for preserving ovarian function during aging. To solve this problem, we developed a long-term treatment for human embryonic stem cell-derived mesenchymal progenitor cells (hESC-MPCs). We investigated whether the cells retained their ability to resist ovarian aging, which leads to delayed reproductive senescence. MATERIALS AND METHODS: In a middle-aged female model undergoing natural aging, we analyzed whether hESC-MPCs benefit the long-term maintenance of reproductive fecundity and ovarian reservoirs and how their transplantation regulates ovarian function. RESULTS: The number of primordial follicles and mice with regular estrous cycles were increased in perimenopausal mice who underwent multiple introductions of hESC-MPCs compared to age-matched controls. The estradiol levels in the hESC-MPCs group were restored to those in the young and adult groups. Embryonic development and live birth rates were higher in the hESC-MPC group than in the control group, suggesting that hESC-MPCs delayed ovarian senescence. In addition to their direct effects on the ovary, multiple-treatments with hESC-MPCs reduced ovarian fibrosis by downregulating inflammation and fibrosis-related genes via the suppression of myeloid-derived suppressor cells (MDSCs) produced in the bone marrow. CONCLUSIONS: Multiple introductions of hESC-MPCs could be a useful approach to prevent female reproductive senescence and that these cells are promising sources for cell therapy to postpone the ovarian aging and retain fecundity in perimenopausal women.

The aging ovary impairs acute stroke outcomes.

In experimental stroke, ovariectomized (OVX) adult rats have larger infarct volumes and greater sensory-motor impairment as compared to ovary-intact females and is usually interpreted to indicate that ovarian hormones are neuroprotective for stroke. Previous work from our lab shows that middle-aged, acyclic reproductively senescent (RS) females have worse stroke outcomes as compared to adult (normally cycling) females. We hypothesized that if loss of ovarian estrogen is the critical determinant of stroke outcomes, then ovary-intact middle-aged acyclic females, who have reduced levels of estradiol, should have similar stroke outcomes as age-matched OVX. Instead, the data demonstrated that OVX RS animals showed better sensory-motor function after stroke and reduced infarct volume as compared to ovary-intact females. Inflammatory cytokines were decreased in the aging ovary after stroke as compared to non-stroke shams, which led to the hypothesis that immune cells may be extravasated from the ovaries post-stroke. Flow cytometry indicated reduced overall T cell populations in the aging ovary after middle cerebral artery occlusion (MCAo), with a paradoxical increase in regulatory T cells (Tregs) and M2-like macrophages. Moreover, in the brain, OVX RS animals showed increased Tregs, increased M2-like macrophages, and increased MHC II + cells as compared to intact RS animals, which have all been shown to be correlated with better prognosis after stroke. Depletion of ovary-resident immune cells after stroke suggests that there may be an exaggerated response to ischemia and possible increased burden of the inflammatory response via extravasation of these cells into circulation. Increased anti-inflammatory cells in the brain of OVX RS animals further supports this hypothesis. These data suggest that stroke severity in aging females may be exacerbated by the aging ovary and underscore the need to assess immunological changes in this organ after stroke.

Vitellogenin accumulation leads to reproductive senescence by impairing lysosomal function.

The maintenance of proteostasis is essential for cellular and organism healthspan. How proteostasis collapse influences reproductive span remains largely unclear. In Caenorhabditis elegans, excess accumulation of vitellogenins, the major components in yolk proteins, is crucial for the development of the embryo and occurs throughout the whole body during the aging process. Here, we show that vitellogenin accumulation leads to reproduction cessation. Excess vitellogenin is accumulated in the intestine and transported into the germline, impairing lysosomal activity in these tissues. The lysosomal function in the germline is required for reproductive span by maintaining oocyte quality. In contrast, autophagy and sperm depletion are not involved in vitellogenin accumulation-induced reproductive aging. Our findings provide insights into how proteome imbalance has an impact on reproductive aging and imply that improvement of lysosomal function is an effective approach for mid-life intervention for maintaining reproductive health in mammals.

IGF1 gene therapy in middle-aged female rats delays reproductive senescence through its effects on hypothalamic GnRH and kisspeptin neurons.

The process of aging is the result of progressive loss of homeostasis and functional body impairment, including the central nervous system, where the hypothalamus plays a key role in regulating aging mechanisms. The consequences of aging include a chronic proinflammatory environment in the hypothalamus that leads to decreased secretion of gonadotropin-releasing hormone (GnRH) and impairs kisspeptin neuron functionality. In this work, we investigated the effect of insulin-like growth factor 1 (IGF1) gene therapy on hypothalamic kisspeptin/GnRH neurons and on microglial cells, that mediate the inflammatory process related with the aging process. The results show that IGF1 rats have higher kisspeptin expression in the anteroventral periventricular (AVPV) nucleus and higher immunoreactivity of GnRH in the arcuate nucleus and median eminence. In addition, IGF1-treated animals exhibit increased numbers of Iba1+ microglial cells and MHCII+/Iba1+ in the AVPV and arcuate nuclei. In conclusion, IGF1 gene therapy maintains kisspeptin production in the AVPV nucleus, induces GnRH release in the median eminence, and alters the number and reactivity of microglial cells in middle-aged female rats. We suggest that IGF1 gene therapy may have a protective effect against reproductive decline.

Central and peripheral neuropeptide RFRP-3: A bridge linking reproduction, nutrition, and stress response.

This article is an amalgamation of the current status of RFRP-3 (GnIH) in reproduction and its association with the nutrition and stress-mediated changes in the reproductive activities. GnIH has been demonstrated in the hypothalamus of all the vertebrates studied so far and is a well-known inhibitor of GnRH mediated reproduction. The RFRP-3 neurons interact with the other hypothalamic neurons and the hormonal signals from peripheral organs for coordinating the nutritional, stress, and environmental associated changes to regulate reproduction. RFRP-3 has also been shown to regulate puberty, reproductive cyclicity and senescence depending upon the nutritional status. A favourable nutritional status and the environmental cues which are permissive for the successful breeding and pregnancy outcome keep RFRP-3 level low, whereas unfavourable nutritional status and stressful conditions increase the expression of RFRP-3 which impairs the reproduction. Still our knowledge about RFRP-3 is incomplete regarding its therapeutic application for nutritional or stress-related reproductive disorders.

Age-dependent change of RFRP-3 neuron numbers and innervation in female mice.

In female mammals, reproductive senescence is a complex process involving progressive ovarian dysfunction, associated with altered central control of the hypothalamic-pituitary-gonadal axis and desynchronization of the circadian system. The objective of this study was to investigate age-dependent changes in the daily regulation of Arg-Phe amide-related peptide-3 (RFRP-3), a hypothalamic peptide involved in reproduction, in female C57BL/6 J mice of different age groups (4, 13, and 19 months old) sampled at their diestrus stage. We found an age-dependent decrease in the total number of RFRP-3 neurons and in the relative number of activated (i.e. c-Fos-positive) RFRP-3 neurons. RFRP-3 neuronal activation exhibited a daily variation in young and middle-aged mice, which was abolished in 19-month-old mice. We also found a daily variation in the number of RFRP-3 neurons receiving close vasopressin (AVP)- and vasoactive intestinal peptide (VIP)-ergic fiber appositions in mice aged 4 and 13 months, but not in 19-month-old mice. However, we found no daily or age-dependent changes in the AVP and VIP fiber density in the dorsomedial hypothalamus. Plasma LH levels were similar in mice aged 4 and 13 months, but were markedly increased in 19-month-old mice. The present findings indicate that the number of RFRP-3 positive neurons is downregulated during old age and that the daily changes in their innervation by the circadian peptides AVP and VIP are abolished. This age-associated reduced (rhythmic) activity of the inhibitory RFRP-3 system could be implicated in the elevated LH secretion observed during reproductive senescence.

Cyclophosphamide, a cancer chemotherapy drug-induced early onset of reproductive senescence and alterations in reproductive performance and their prevention in mice.

Although, cyclophosphamide (CP) treatment is known to cause degeneration of the ovarian follicular reserve, which may have a serious consequence of the onset of early reproductive senescence, thus far there is no experimental study either to demonstrate CP-induced early onset of reproductive senescence or its prevention. Intraperitoneal administration (ip) of CP [100 mg/kg body weight (bw)/mouse] resulted in a drastic reduction in reproductive life span as shown by the onset of reproductive senescence at a significantly early age (258 days) compared to controls (349 days), whereas treatment with the root extract of the herb Decalepis hamiltonii (DH) (200 mg/Kg bw/day for 7 days), a cocktail of anti-oxidants prior to CP administration maintained normal reproductive life span in mice. Further, the CP treated mice showed a significant increase in pre-coital interval and a significant reduction in parturition index coupled with regressive changes in the uterine endometrium, whereas DH co-treatment prevented these changes. The results for the first time, demonstrate that the ovarian toxicity of CP could be prevented by an anti-oxidant to maintain a normal reproductive life span as well as reproductive outcome using mice model.

Disarranged neuroplastin environment upon aging and chronic stress recovery in female Sprague Dawley rats.

Chronic stress produces long-term metabolic changes throughout the superfamily of nuclear receptors, potentially causing various pathologies. Sex hormones modulate the stress response and generate a sex-specific age-dependent metabolic imprint, especially distinct in the reproductive senescence of females. We monitored chronic stress recovery in two age groups of female Sprague Dawley rats to determine whether stress and/or aging structurally changed the glycolipid microenvironment, a milieu playing an important role in cognitive functions. Old females experienced memory impairment even at basal conditions, which was additionally amplified by stress. On the other hand, the memory of young females was not disrupted. Stress recovery was followed by a microglial decrease and an increase in astrocyte count in the hippocampal immune system. Since dysfunction of the brain immune system could contribute to disturbed synaptogenesis, we analyzed neuroplastin expression and the lipid environment. Neuroplastin microenvironments were explored by analyzing immunofluorescent stainings using a newly developed Python script method. Stress reorganized glycolipid microenvironment in the Cornu Ammonis 1 (CA1) and dentate gyrus (DG) hippocampal regions of old females but in a very different fashion, thus affecting neuroplasticity. The postulation of four possible neuroplastin environments pointed to the GD1a ganglioside enrichment during reproductive senescence of stressed females, as well as its high dispersion in both regions and to GD1a and GM1 loss in the CA1 region. A specific lipid environment might influence neuroplastin functionality and underlie synaptic dysfunction triggered by a combination of aging and chronic stress.

Disentangling the effects of male age and mating history: Contrasting effects of mating history on precopulatory mating behavior and paternity success.

Many studies ask whether young or older males are better at acquiring mates. Even so, how age affects reproductive success is still poorly understood because male age and mating history are confounded in most studies: older males usually have more mating experience. To what extent does mating history rather than age explain variation in male mating success? And how do mating history and male age determine paternity when there is also postcopulatory sexual selection? Here, we experimentally manipulated the mating history of old and young males in the eastern mosquitofish (Gambusia holbrooki). We then recorded male mating behavior and share of paternity (1259 offspring from 232 potential sires) when they competed for mates and fertilizations. Old males, and males with no mating experience, spent significantly more time approaching females, and attempting to mate, than did young males and those with greater mating experience. Male age and mating history interacted to affect paternity: old males benefited from having previous mating experience, but young males did not. Our results highlight that the age-related changes in male reproductive traits and in paternity that have been described in many taxa may be partly attributable to male mating history and not simply to age itself.

Should females prefer old males?

Whether females should prefer to mate with old males is controversial. Old males may sire offspring of low quality because of an aging germline, but their proven ability to reach an old age can also be an excellent indicator of superior genetic quality, especially in natural populations. These genetic effects are, however, hard to study in nature, because they are often confounded with direct benefits offered by old males to the female, such as experience and high territory quality. We, therefore, used naturally occurring extra-pair young to disentangle different aspects of male age on female fitness in a natural population of collared flycatchers because any difference between within- and extra-pair young within a nest should be caused by paternal genetic effects only. Based on 18 years of long-term data, we found that females paired with older males as social partners experienced an overall reproductive advantage. However, offspring sired by old males were of lower quality as compared to their extra-pair half-siblings, whereas the opposite was found in nests attended by young males. These results imply a negative genetic effect of old paternal age, given that extra-pair males are competitive middle-age males. Thus, offspring may benefit from being sired by young males but raised by old males, to maximize both genetic and direct effects. Our results show that direct and genetic benefits from pairing with old males may act in opposing directions and that the quality of the germline may deteriorate before other signs of senescence become obvious.

Individual telomere dynamics and their links to life history in a viviparous lizard.

Emerging patterns suggest telomere dynamics and life history are fundamentally linked in endotherms through life-history traits that mediate the processes underlying telomere attrition. Unlike endotherms, ectotherms maintain the ability to lengthen somatic telomeres throughout life and the link between life-history strategies and ectotherm telomere dynamics is unknown. In a well-characterized model system (Niveoscincus ocellatus), we used long-term longitudinal data to study telomere dynamics across climatically divergent populations. We found longer telomeres in individuals from the cool highlands than those from the warm lowlands at birth and as adults. The key determinant of adult telomere length across populations was telomere length at birth, with population-specific effects of age and growth on adult telomere length. The reproductive effort had no proximate effect on telomere length in either population. Maternal factors influenced telomere length at birth in the warm lowlands but not the cool highlands. Our results demonstrate that life-history traits can have pervasive and context-dependent effects on telomere dynamics in ectotherms both within and between populations. We argue that these telomere dynamics may reflect the populations' different life histories, with the slow-growing cool highland population investing more into telomere lengthening compared to the earlier-maturing warm lowland population.

1,25-Dihydroxyvitamin D deficiency accelerates male reproductive senescence in aging mice and 1,25(OH)2D3 alleviates oxidative stress via NF-κB/SOD.

1,25(OH)2D3 has been demonstrated to exert direct actions on male reproductive system in humans or in animals. With age, renal synthesis of 1,25(OH)2D3 declines significantly, and vitamin D supplementation has been found to alleviate the manifestations of male reproductive aging. Therefore, the relationship between 1,25(OH)2D3 and male reproductive aging needs further study. To determine whether 1,25(OH)2D3 deficiency accelerates male reproductive senescence in aging mice, wild-type and 1α(OH)ase-/- male mice fed a rescue diet after weaning, and the reproductive phenotypes were evaluated at 12-18 mo of age. We demonstrated that 1,25(OH)2D3 deficiency accelerated male reproductive senescence, representing lower fertility efficiency and gonadal hormone levels, reducing cell proliferation, and increasing cell apoptosis, cellular senescence, and the senescence-associated secretory phenotype (SASP). We confirmed that the increased oxidative stress and DNA damage detected in 1α(OH)ase-/- mice resulted in accelerated reproductive senescence in reproductive system, since exogenous antioxidant pyrroloquinoline quinone (PQQ) supplementation could largely rescue reproductive aging phenotype. We further validated the antioxidant effect of 1,25(OH)2D3 in aging wild-type mice and senescent Leydig cells by treated 18-mo-old wild-type male mice or TM3 cells with 1,25(OH)2D3 or vehicle. We assessed the differential gene expression between grouped senescent TM3 cells using RNA-Seq and verified 1,25(OH)2D3 exerted an antioxidant role by acting NF-κB/SOD. This study suggests that 1,25(OH)2D3 deficiency accelerates male reproductive senescence in aging mice by increasing oxidative stress and 1,25(OH)2D3 plays a role in alleviating oxidative stress via NF-κB/SOD signaling pathway.NEW & NOTEWORTHY Based on this studies, we propose that 1,25(OH)2D3 can delay male reproductive aging, and we also propose that 1,25(OH)2D3 regulates NF-κB to exert antioxidant effect. Therefore, by targeting a fundamental aging mechanism, 1,25(OH)2D3 may be an effective agent in maintaining fertility and postponing male reproductive senescence.

Paternal age negatively affects sperm production of the progeny.

Parental age has profound consequences for offspring's phenotype. However, whether patrilineal age affects offspring sperm production remains unknown, despite the importance of sperm production for male reproductive success in species facing post-copulatory sexual selection. Using a longitudinal dataset on ejaculate attributes of the houbara bustard, we showed that offspring sired by old fathers had different age-dependent trajectories of sperm production compared to offspring sired by young fathers. Specifically, they produced less sperm (-48%) in their first year of life, and 14% less during their lifetime. Paternal age had the strongest effect, with weak evidence for grandpaternal or great grandpaternal age effects. These results show that paternal age can affect offspring reproductive success by reducing sperm production, establishing an intergenerational link between ageing and sexual selection.

Loss of α-gal during primate evolution enhanced antibody-effector function and resistance to bacterial sepsis.

Most mammals express a functional GGTA1 gene encoding the N-acetyllactosaminide α-1,3-galactosyltransferase enzyme, which synthesizes Gal-α1-3Gal-ß1-4GlcNAc (α-gal) and are thus tolerant to this self-expressed glycan. Old World primates including humans, however, carry loss-of-function mutations in GGTA1 and lack α-gal. Presumably, fixation of such mutations was propelled by natural selection, favoring the emergence of α-gal-specific immunity, conferring resistance to α-gal-expressing pathogens. Here, we show that loss of Ggta1 function in mice enhances resistance to bacterial sepsis, irrespectively of α-Gal-specific immunity. Rather, the absence of α-gal from IgG-associated glycans increases IgG effector function via a mechanism associated with enhanced IgG-Fc gamma receptor (FcγR) binding. The ensuing survival advantage against sepsis comes alongside a cost of accelerated reproductive senescence in Ggta1-deleted mice. Mathematical modeling of this trade-off suggests that high exposure to virulent pathogens exerts sufficient selective pressure to fix GGTA1 loss-of-function mutations, as likely occurred during the evolution of primates toward humans.

Temporal dynamics of competitive fertilization in social groups of red junglefowl (Gallus gallus) shed new light on avian sperm competition.

Studies of birds have made a fundamental contribution to elucidating sperm competition processes, experimentally demonstrating the role of individual mechanisms in competitive fertilization. However, the relative importance of these mechanisms and the way in which they interact under natural conditions remain largely unexplored. Here, we conduct a detailed behavioural study of freely mating replicate groups of red junglefowl, Gallus gallus, to predict the probability that competing males fertilize individual eggs over the course of 10-day trials. Remating frequently with a female and mating last increased a male's probability of fertilization, but only for eggs ovulated in the last days of a trial. Conversely, older males, and those mating with more polyandrous females, had consistently lower fertilization success. Similarly, resistance to a male's mating attempts, particularly by younger females, reduced fertilization probability. After considering these factors, male social status, partner relatedness and the estimated state of male extragonadal sperm reserves did not predict sperm competition outcomes. These results shed new light on sperm competition dynamics in taxa such as birds, with prolonged female sperm storage and staggered fertilizations. This article is part of the theme issue 'Fifty years of sperm competition'.

The hidden ageing costs of sperm competition.

Ageing and sexual selection are intimately linked. There is by now compelling evidence from studies performed across diverse organisms that males allocating resources to mating competition incur substantial physiological costs, ultimately increasing ageing. However, although insightful, we argue here that to date these studies cover only part of the relationship linking sexual selection and ageing. Crucially, allocation to traits important in post-copulatory sexual selection, that is sperm competition, has been largely ignored. As we demonstrate, such allocation could potentially explain much diversity in male and female ageing patterns observed both within and among species. We first review how allocation to sperm competition traits such as sperm and seminal fluid production depends on the quality of resources available to males and can be associated with a wide range of deleterious effects affecting both somatic tissues and the germline, and thus modulate ageing in both survival and reproductive terms. We further hypothesise that common biological features such as plasticity, prudent sperm allocation and seasonality of ejaculate traits might have evolved as counter-adaptations to limit the ageing costs of sperm competition. Finally, we discuss the implications of these emerging ageing costs of sperm competition for current research on the evolutionary ecology of ageing.

Decreased Anti-Müllerian hormone and Anti-Müllerian hormone receptor type 2 in hypothalami of old Japanese Black cows.

Cow fertility decreases with age, but the hypothalamic pathomechanisms are not understood. Anti-Müllerian hormone (AMH) stimulates gonadotropin-releasing hormone (GnRH) neurons via AMH receptor type 2 (AMHR2), and most GnRH neurons in the preoptic area (POA), arcuate nucleus (ARC), and median eminence (ME) express AMH and AMHR2. Therefore, we hypothesized that both protein amounts would differ in the anterior hypothalamus (containing the POA) and posterior hypothalamus (containing the ARC and ME) between young post-pubertal heifers and old cows. Western blot analysis showed lower (P<0.05) expressions of AMH and AMHR2 in the posterior hypothalamus, but not in the anterior hypothalamus, of old Japanese Black cows compared to young heifers. Therefore, AMH and AMHR2 were decreased in the posterior hypothalami of old cows.

Methods for Assessing Fertility in C. elegans from a Single Population.

Reproductive senescence occurs in a wide range of species with mechanistic aspects that are conserved from Caenorhabditis elegans to humans. Genetic and environmental factors can influence fertility and reproductive output can impact rates of aging. The C. elegans Bristol N2 strain commonly used in laboratories is hermaphroditic, producing a defined number of sperm during larval development before switching exclusively to oogenesis. Here we show a method of assaying both oocyte and sperm quality from a single population of animals.

Post-copulatory sexual selection allows females to alleviate the fitness costs incurred when mating with senescing males.

Male senescence has detrimental effects on reproductive success and offspring fitness. When females mate with multiple males during the same reproductive bout, post-copulatory sexual selection that operates either through sperm competition or cryptic female choice might allow females to skew fertilization success towards young males and as such limit the fitness costs incurred when eggs are fertilized by senescing males. Here, we experimentally tested this hypothesis. We artificially inseminated female North African houbara bustards with sperm from dyads of males of different (young and old) or similar ages (either young or old). Then, we assessed whether siring success was biased towards young males and we measured several life-history traits of the progeny to evaluate the fitness costs due to advanced paternal age. In agreement with the prediction, we found that siring success was biased towards young males, and offspring sired by old males had impaired hatching success, growth and post-release survival (in females). Overall, our results support the hypothesis that post-copulatory sexual selection might represent an effective mechanism allowing females to avoid the fitness costs of fertilization by senescing partners.