Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

Oocyte quality is enhanced by hypoglycosylated FSH through increased cell-to-cell interaction during mouse follicle development.

Macroheterogeneity in follicle-stimulating hormone (FSH) ß-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

KAT8 functions in redox homeostasis and mitochondrial dynamics during mouse oocyte meiosis progression.

As a histone acetyltransferase, lysine acetyltransferase 8 (KAT8) participates in diverse biological processes. However, the effect of KAT8 on oocyte maturation in mice remains unclear. In this study, we found that mouse oocytes overexpressing Kat8-OE induced maturation failure manifested reduced rates of GVBD and first polar body emission. In addition, immunostaining results revealed that Kat8 overexpressing oocytes showed inappropriate mitochondrial distribution patterns, overproduction of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX, hyperacetylation of α-tubulin, and severely disrupted spindle/chromosome organization. Moreover, we revealed that Kat8 overexpression induced a decline in SOD1 proteins and KAT8's interaction with SOD1 in mouse ovaries via immunoprecipitation. Western blotting data confirmed that Kat8-OE induced downregulation of SOD1 expression, which is a key factor for the decline of oocyte quality in advanced maternal age. Also, the injection of Myc-Sod1 cRNA could partially rescue maternal age-induced meiotic defects in oocytes. In conclusion, our data demonstrated that high level of KAT8 inhibited SOD1 activity, which in turn induced defects of mitochondrial dynamics, imbalance of redox homeostasis, and spindle/chromosome disorganization during mouse oocyte maturation.

Oocytes orchestrate protein prenylation for mitochondrial function through selective inactivation of cholesterol biosynthesis in murine species.

Emerging research and clinical evidence suggest that the metabolic activity of oocytes may play a pivotal role in reproductive anomalies. However, the intrinsic mechanisms governing oocyte development regulated by metabolic enzymes remain largely unknown. Our investigation demonstrates that geranylgeranyl diphosphate synthase1 (Ggps1), the crucial enzyme in the mevalonate pathway responsible for synthesizing isoprenoid metabolite geranylgeranyl pyrophosphate from farnesyl pyrophosphate, is essential for oocyte maturation in mice. Our findings reveal that the deletion of Ggps1 that prevents protein prenylation in fully grown oocytes leads to subfertility and offspring metabolic defects without affecting follicle development. Oocytes that lack Ggps1 exhibit disrupted mitochondrial homeostasis and the mitochondrial defects arising from oocytes are inherited by the fetal offspring. Mechanistically, the excessive farnesylation of mitochondrial ribosome protein, Dap3, and decreased levels of small G proteins mediate the mitochondrial dysfunction induced by Ggps1 deficiency. Additionally, a significant reduction in Ggps1 levels in oocytes is accompanied by offspring defects when females are exposed to a high-cholesterol diet. Collectively, this study establishes that mevalonate pathway-protein prenylation is vital for mitochondrial function in oocyte maturation and provides evidence that the disrupted protein prenylation resulting from an imbalance between farnesyl pyrophosphate and geranylgeranyl pyrophosphate is the major mechanism underlying impairment of oocyte quality induced by high cholesterol.

Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Molecular Mechanisms Determining Mammalian Oocyte Quality with the Treatment of Cancer Therapy.

Cancer is a global public health issue and remains one of the leading causes of death in the United States (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). It is estimated in the US in 2022, about 935,000 new cases of cancer will be diagnosed in women, and the probability of developing invasive cancer is 5.8% for females younger than 50 years old (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). However, advances in screening programs, diagnostic methods, and therapeutic options have greatly increased the five-year survival rate in reproductive-age women with a variety of cancers. Given the clinical consequences of gonadotoxic cancer therapies, young, female cancer survivors may face compromised fertility, premature ovarian insufficiency, early-onset menopause, and endocrine dysregulation (Bedoschi et al. Future Oncol. 12:2333-44, 2016). Gonadotoxic side effects may include decreased oocyte quality within surviving follicles, loss of ovarian follicles, and impaired ovarian function. In reproductive-age women, oocyte quality is an important element for successful clinical pregnancies and healthy offspring as poor-quality oocytes may be a cause of infertility (McClam et al. Biol Reprod. 106:328-37, 2022; Marteil et al. Reprod Biol. 9:203-24, 2009; Krisher. J Anim Sci. 82: E14-E23, 2004). Thus, it is critical to determine the quantity and quality of surviving follicles in the ovary after cancer treatment and to assess oocyte quality within those surviving follicles as these are markers for determining the capacity for ovarian function restoration and future fertility, especially for young cancer survivors (Xu et al. Nat Med. 17:1562-3, 2011). The long-term effects of cancer therapeutics on oocyte quality are influenced by factors including, but not limited to, individual patient characteristics (e.g. age, health history, comorbidities, etc.), disease type, or treatment regimen (Marci et al. Reprod Biol Endocrinol. 16:1-112, 2018). These effects may translate clinically into an impaired production of viable oocytes and compromised fertility (Garutti et al. ESMO Open. 6:100276, 2021).

Role of FBXL5 in redox homeostasis and spindle assembly during oocyte maturation in mice.

As an E3 ubiquitin ligase, F-box and leucine-rich repeat protein 5 (FBXL5) participates in diverse biologic processes. However, the role of Fbxl5 in mouse oocyte meiotic maturation has not yet been fully elucidated. The present study revealed that mouse oocytes depleted of Fbxl5 were unable to complete meiosis, as Fbxl5 silencing led to oocyte meiotic failure with reduced rates of GVBD and polar body extrusion. In addition, Fbxl5 depletion induced aberrant mitochondrial dynamics as we noted the overproduction of reactive oxygen species (ROS) and the accumulation of phosphorylated γH2AX with Fbxl5 knockdown. We also found that Fbxl5-KD led to the abnormal accumulation of CITED2 proteins in mouse oocytes. Our in vitro ubiquitination assay showed that FBXL5 interacted with CITED2 and that it mediated the degradation of CITED2 protein through the ubiquitination-proteasome pathway. Collectively, our data revealed critical functions of FBXL5 in redox hemostasis and spindle assembly during mouse oocyte maturation.

Near-infrared fluorophore IR-61 delays postovulatory aging of mouse oocytes through suppressing oxidative stress mediated by mitochondrial protection.

Postovulatory aging can trigger deterioration of oocyte quality and subsequent embryonic development, and thus reduce the success rates of assisted reproductive technology (ART). The molecular mechanisms underlying postovulatory aging, and preventative strategies, remain to be explored. The near-infrared fluorophore IR-61, a novel heptamethine cyanine dye, has the potential for mitochondrial targeting and cell protection. In this study, we found that IR-61 accumulated in oocyte mitochondria and reduced the postovulatory aging-induced decline in mitochondrial function, including mitochondrial distribution, membrane potential, mtDNA number, ATP levels, and mitochondrial ultrastructure. In addition, IR-61 rescued postovulatory aging-caused oocyte fragmentation, defects in spindle structure, and embryonic developmental potential. RNA sequencing analysis indicated that the postovulatory aging-induced oxidative stress pathway might be inhibited by IR-61. We then confirmed that IR-61 decreased the levels of reactive oxygen species and MitoSOX, and increased GSH content in aged oocytes. Collectively, the results indicate that IR-61 may prevent postovulatory aging by rescuing oocyte quality, promoting successful rate in ART procedure.

Indirect markers of oocyte quality in patients with ovarian endometriosis undergoing IVF/ICSI: a systematic review and meta-analysis.

This systematic review and meta-analysis aimed to evaluate the impact of ovarian endometriomas (OMA) on indirect markers of oocyte quality in patients undergoing IVF, compared with women without anatomical or functional ovarian abnormalities. The search spanned original randomized controlled trials, case-control studies and cohort studies published in MEDLINE, the Cochrane Controlled Trials Register and the ClinicalTrials.gov database up to October 2023. Thirty-one studies were included in the meta-analysis, showing no significant differences in fertilization (OR 1.10, 95% CI 0.94-1.30), blastulation (OR 0.86, 95% CI 0.64-1.14) and cancellation (OR 1.06, 95% CI 0.78-1.44) rates. However, patients with OMA exhibited significantly lower numbers of total and mature (metaphase II) oocytes retrieved (mean difference -1.59, 95% CI -2.25 to -0.94; mean difference -1.86, 95% CI -2.46 to -1.26, respectively), and lower numbers of top-quality embryos (mean difference -0.49, 95% CI -0.92 to -0.06). The Ovarian Sensitivity Index was similar between the groups (mean difference -1.55, 95% CI -3.27 to 0.18). The lack of data published to date prevented meta-analysis on euploidy rate. In conclusion, although the presence of OMA could decrease the oocyte yield in patients undergoing IVF/intracytoplasmic sperm injection, it does not appear to have an adverse impact on oocyte quality.

Long non-coding RNA PWRN1 affects ovarian follicular development by regulating the function of granulosa cells.

RESEARCH QUESTION: What is the role of Prader-Willi region non-protein coding RNA 1 (PWRN1) in ovarian follicular development and its molecular mechanism? DESIGN: The expression and localization of PWRN1 were detected in granulosa cells from patients with different ovarian functions, and the effect of interfering with PWRN1 expression on cell function was detected by culturing granulosa cells in vitro. Furthermore, the effects of interfering with PWRN1 expression on ovarian function of female mice were explored through in-vitro and in-vivo experiments. RESULTS: The expression of PWRN1 was significantly lower in granulosa cells derived from patients with diminished ovarian reserve (DOR) compared with patients with normal ovarian function. By in-vitro culturing of primary granulosa cells or the KGN cell line, the results showed that the downregulation of PWRN1 promoted granulosa cell apoptosis, caused cell cycle arrested in S-phase, generated high levels of autophagy and led to significant decrease in steroidogenic capacity, including inhibition of oestradiol and progesterone production. In addition, SIRT1 overexpression could partially reverse the inhibitory effect of PWRN1 downregulation on cell proliferation. The results of in-vitro culturing of newborn mouse ovary showed that the downregulation of PWRN1 could slow down the early follicular development. Further, by injecting AAV-sh-PWRN1 in mouse ovarian bursa, the oestrous cycle of mouse was affected, and the number of oocytes retrieved after ovulation induction and embryos implanted after mating was significantly reduced. CONCLUSION: This study systematically elucidated the novel mechanism by which lncRNA PWRN1 participates in the regulation of granulosa cell function and follicular development.