Ulrike Luderer

Over 25% of active NASA astronauts are women who will be exposed to low daily doses and dose rates of galactic cosmic rays (GCR) in space. We hypothesized that exposing mice to a preliminary simulated GCR mixed heavy ion beam composed of iron, silicon, and titanium ions induces follicle depletion and dose-dependent ovarian tumors. Female mice were exposed to 10, or 20 cGy each of Fe, Si, and Ti ions or sham-irradiation in quick succession within 15 min for total doses of 0, 30, or 60 cGy of the three beams. 16 months later, their ovaries were removed. Hyperplasia of the ovarian surface epithelium (OSE) was noted in 13%, 59%, and 22% of the 0, 30, and 60 cGy irradiated mice, respectively. The prevalence of mixed ovarian tumors was 0, 6, and 89%, respectively, in the 0, 30, and 60 cGy groups. Low numbers of Ki67 positive OSE and tumor cells supported a benign tumor phenotype. In a separate study, Si ion irradiation alone at 32 cGy did not induce ovarian tumors in mice; however, the mixed heavy ions at all doses and Si ion irradiation alone reduced the total number of healthy ovarian follicles. Mixed heavy ion exposure reduced lipid peroxidation, fibrosis, inflammation, and lipofuscin accumulation at 60 cGy compared to 0 cGy, but elevated inflammation and lipofuscin accumulation at 30 cGy compared to 60 cGy. Preliminary simulated GCR exposure causes ovarian follicle death and tumorigenesis. This study provides insight into space-radiation induced ovarian damage and cancer risk in females.

Cannabis use typically starts in early to mid-adolescence. Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis, targets cannabinoid receptors (CBRs) to exert its pharmacological effects. Expression of CBRs has been observed in human and rodent testes, but their potential role in the control of reproductive function remains unclear. We aimed to elucidate how THC exposure during adolescence or young adulthood affects the reproductive health of males. C57BL/6N male mice were given THC (5 mg/kg) or vehicle, once daily by intraperitoneal injection from postnatal day (PND) 30 to PND 43 (adolescent exposure) or PND 70 to PND 83 (adult exposure), and testes were harvested at PND 70 and PND 110, respectively. Results showed that CBRs (CB1R and CB2R) and enzymes that biosynthesize or inactivate the endocannabinoids anandamide–N-acylphosphatidylethanolamine phospholipase D or fatty acid amide hydrolase, respectively—are expressed in the mouse testis. THC exposure in adolescence decreased sperm numbers and increased seminiferous tubule degeneration in young adult testes, whereas adult exposure did not affect spermatogenesis and seminiferous tubule morphology. Both adolescent and adult THC exposure resulted in decreased plasma testosterone levels; however, only mice with adolescent THC exposure showed impaired steroidogenesis with dysregulated expression of steroidogenic acute regulatory protein and steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A1). Our results support that adolescent THC exposure may cause testicular toxicity through direct and aberrant activation of CBRs in the testis. These studies show that the adolescent testis is more sensitive than the adult testis to THC-induced disruption of spermatogenesis.

In brief: Particulate matter (PM) air pollution contains many chemicals that are individually known to have adverse effects on reproduction, as well as numerous others that have not been investigated for effects on reproduction. This review discusses the scientific literature investigating the effects of ambient and experimentally generated PM mixtures on reproduction. PM air pollution consists of liquid and solid particles, which are categorized by size as less than 10 (PM ₁₀ ) μm, 2.5 (PM _2.5 ) μm, or 0.1 μm (PM _0.1 or ultrafine) in aerodynamic diameter and which vary in composition depending on the sources. PM exposure is ubiquitous and has been associated with many adverse health effects. This narrative review focuses on epidemiological and experimental studies that investigated the effects of PM exposure on female and male reproduction and on pregnancy.

Prior studies showed that mice deficient in the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in synthesis of the thiol antioxidant glutathione (GSH), have decreased ovarian GSH concentrations, chronic ovarian oxidative stress, poor oocyte quality resulting in early preimplantation embryonic mortality and decreased litter size, and accelerated age-related decline in ovarian follicle numbers. Global deficiency of the catalytic subunit of this enzyme, Gclc, is embryonic lethal. We tested the hypothesis that granulosa cell- or oocyte-specific deletion of Gclc recapitulates the female reproductive phenotype of global Gclm deficiency. We deleted Gclc in granulosa cells or oocytes of growing follicles using Gclc floxed transgenic mice paired with Amhr2-Cre or Zp3-Cre alleles respectively. We discovered that granulosa cell-specific deletion of Gclc in Amhr2Cre;Gclc(f/−) mice recapitulates the decreased litter size observed in Gclm−/− mice, but does not recapitulate the accelerated age-related decline in ovarian follicles observed in Gclm−/− mice. In addition to having lower GSH concentrations in granulosa cells, Amhr2Cre;Gclc(f/−) mice also had decreased GSH concentrations in oocytes. By contrast, oocyte-specific deletion of Gclc in Zp3Cre;Gclc(f/−) mice did not affect litter size or accelerate the age-related decline in follicle numbers, and these mice did not have decreased oocyte GSH concentrations, consistent with transport of GSH between cells via gap junctions. The results suggest that GSH deficiency at earlier stages of follicle development may be required to generate the accelerated follicle depletion phenotype observed in global Gclm null mice.

People are widely exposed to polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP). Prior studies showed that prenatal exposure to BaP depletes germ cells in ovaries, causing earlier onset of ovarian senescence post-natally; developing testes were affected at higher doses than ovaries. Our primary objective was to determine if prenatal BaP exposure results in transgenerational effects on ovaries and testes. We orally dosed pregnant germ cell-specific EGFP-expressing mice (F0) with 0.033, 0.2, or 2 mg/kg-day BaP or vehicle from embryonic day (E) 6.5–11.5 (F1 offspring) or E6.5–15.5 (F2 and F3). Ovarian germ cells at E13.5 and follicle numbers at postnatal day 21 were significantly decreased in F3 females at all doses of BaP; testicular germ cell numbers were not affected. E13.5 germ cell RNA-sequencing revealed significantly increased expression of male-specific genes in female germ cells across generations and BaP doses. Next, we compared the ovarian effects of 2 mg/kg-day BaP dosing to wild type C57BL/6J F0 dams from E6.5–11.5 or E12.5–17.5. We observed no effects on F3 ovarian follicle numbers with either of the shorter dosing windows. Our results demonstrate that F0 BaP exposure from E6.5–15.5 decreased the number of and partially disrupted transcriptomic sexual identity of female germ cells transgenerationally.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ⁹-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30–33 and their ovaries were harvested PND34; other mice received THC from PND30–43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are products of incomplete combustion. In female mouse embryos primordial germ cells proliferate before and after arriving at the gonadal ridge around embryonic (E) 10 and begin entering meiosis at E13.5. Now oocytes, they arrest in the first meiotic prophase beginning at E17.5. We previously reported dose-dependent depletion of ovarian follicles in female mice exposed to 2 or 10 mg/kg-day BaP E6.5–15.5. We hypothesized that embryonic ovaries are more sensitive to gestational BaP exposure during the mitotic developmental window, and that this exposure results in persistent oxidative stress in ovaries and oocytes of exposed F1 female offspring. We orally dosed timed-pregnant female mice with 0 or 2 mg/kg-day BaP in oil from E6.5–11.5 (mitotic window) or E12.5–17.5 (meiotic window). Cultured E13.5 ovaries were utilized to investigate the mechanism of BaP-induced germ cell death. We observed statistically significant follicle depletion and increased ovarian lipid peroxidation in F1 pubertal ovaries following BaP exposure during either prenatal window. Culture of E13.5 ovaries with BaP induced germ cell DNA damage and release of cytochrome c from the mitochondria in oocytes, confirming that BaP exposure induced apoptosis via the mitochondrial pathway. Mitochondrial membrane potential, oocyte lipid droplet (LD) volume, and mitochondrial-LD colocalization were decreased and mitochondrial superoxide levels were increased in the MII oocytes of F1 females exposed gestationally to BaP. Results demonstrate similar sensitivity to germ cell depletion and persistent oxidative stress in F1 ovaries and oocytes following gestational BaP exposure during mitotic or meiotic windows.

Glutathione (GSH) is a tripeptide thiol antioxidant that has been shown to be important to overall reproductive health. Glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis consists of a catalytic and a modifier (GCLM) subunit. We previously showed that oxidative stress in the ovary and oocytes of Gclm^−/− mice is associated with accelerated age-related decline in ovarian follicles and decreased female fertility due to preimplantation embryonic mortality. Mammalian preimplantation development is a highly regulated and energy-intensive process that primarily relies on coordination between lipid droplets (LDs) and mitochondria to maintain cellular homeostasis. In this study, we hypothesized that GSH deficiency in oocytes increases oxidative stress, leading to increased mitochondrial dysfunction and decreased LD consumption, thereby decreasing oocyte developmental competence. We observed that Gclm^−/− oocytes have increased oxidative stress, primarily in the form of mitochondrial superoxide and decreased subcortical mitochondrial clusters. Further, Gclm^−/− oocytes have decreased mitochondrial membrane potential (ΔΨm) compared with Gclm^+/+. We surmise this is likely due to the decreased availability of LDs, as we observed a significant decrease in LD content in Gclm^−/− oocytes compared with Gclm^+/+. The decreased oocyte LD content is likely related to an altered serum lipidome, with Gclm^−/− serum having relatively lower unsaturated fatty acids and triglycerides than that of Gclm^+/+ and Gclm^+/− females. Altogether these data support that decreased LDs and increased oxidative stress are primary drivers of decreased oocyte developmental competence in GSH-deficient oocytes.

Background: Fine particulate matter (PM_2.5) exposure accelerates atherosclerosis and contains known ovotoxic chemicals. However, effects of exposure to PM_2.5 on the finite ovarian follicle pool have hardly been investigated, nor have interactions between ovarian and cardiovascular effects. We hypothesized that subchronic inhalation exposure to human-relevant concentrations of PM_2.5 results in destruction of ovarian follicles via apoptosis induction, as well as accelerated recruitment of primordial follicles into the growing pool. Further, we hypothesized that destruction of ovarian follicles enhances the adverse cardiovascular effects of PM_2.5 in females. Results: Hyperlipidemic apolipoprotein E (Apoe) null ovary-intact or ovariectomized female mice and testis-intact male mice were exposed to concentrated ambient PM_2.5 or filtered air for 12 weeks, 5 days/week for 4 h/day using a versatile aerosol concentration enrichment system. Primordial, primary, and secondary ovarian follicle numbers were decreased by 45%, 40%, and 17%, respectively, in PM_2.5-exposed ovary-intact mice compared to controls (P < 0.05). The percentage of primary follicles with granulosa cells positive for the mitosis marker Ki67 was increased in the ovaries from PM_2.5-exposed females versus controls (P < 0.05), consistent with increased recruitment of primordial follicles into the growing pool. Exposure to PM_2.5 increased the percentages of primary and secondary follicles with DNA damage, assessed by γH2AX immunostaining (P < 0.05). Exposure to PM_2.5 increased the percentages of apoptotic antral follicles, determined by TUNEL and activated caspase 3 immunostaining (P < 0.05). Removal of the ovaries and PM_2.5-exposure exacerbated the atherosclerotic effects of hyperlipidemia in females (P < 0.05). While there were statistically significant changes in blood pressure and heart rate variability in PM_2.5-compared to Air-exposed gonad-intact males and females and ovariectomized females, the changes were not consistent between exposure years and assessment methods. Conclusions: These results demonstrate that subchronic PM_2.5 exposure depletes the ovarian reserve by increasing recruitment of primordial follicles into the growing pool and increasing apoptosis of growing follicles. Further, PM_2.5 exposure and removal of the ovaries each increase atherosclerosis progression in Apoe-/- females. Premature loss of ovarian function is associated with increased risk of osteoporosis, cardiovascular disease and Alzheimer’s disease in women. Our results thus support possible links between PM_2.5 exposure and other adverse health outcomes in women.