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).

KIT in oocytes: a key factor for oocyte survival and reproductive lifespan.

BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).

Sodium thiosulfate does not protect ovarian reserve from cisplatin-induced gonadotoxicity†.

Cisplatin, a platinum-containing alkylating agent, is used in the treatment of various tumors owing to its potent antitumor activity. However, it causes permanent and adverse effects, particularly hearing loss and depletion of ovarian reserve. Until recently, there were no clinically available protective agents to mitigate the adverse side effects of cisplatin-induced cytotoxicity. In 2022, sodium thiosulfate (STS) was approved by the Food and Drug Administration for mitigating hearing loss in children and adolescents undergoing cisplatin treatment. Consequently, our investigation aimed to determine if STS could protect ovarian reserve against cisplatin-induced gonadotoxicity. In an ex vivo culture, the cisplatin-only group exhibited a loss of primordial follicles, while post-STS administration after cisplatin exposure effectively protected primordial follicles. However, when post-STS was administrated either 6 or 4 h after cisplatin exposure, it did not confer protection against cisplatin-induced gonadotoxicity in postnatal day 7 or adolescent mouse models. Immunofluorescence assays using γH2AX and cPARP revealed that oocytes within primordial follicles exhibited DNA damage after cisplatin exposure, irrespective of post-STS administration. This underscores the rapid and heightened sensitivity of oocytes to gonadotoxicity. In addition, oocytes demonstrated an increased expression of pCHK2 rather than pERK, suggesting that the pathway leading to oocyte death differs from the pathway observed in the inner ear cell death following cisplatin exposure. These results imply that while the administration of STS after cisplatin is highly beneficial in preventing hearing loss, it does not confer a protective effect on the ovaries in mouse models.

Metabolic characteristics of granulosa cell tumor: role of PPARγ signaling†.

Granulosa cell tumors are relatively rare, posing challenges for comprehension and therapeutic development due to limited cases and preclinical models. Metabolic reprogramming, a hallmark of cancer, manifests in granulosa cell tumors with notable lipid accumulation and increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), a key lipid metabolism regulator. The roles of these features, however, remain unclear. In our previous work, we established a granulosa cell tumor model in mice by introducing a constitutively active Pik3ca mutant in oocytes, enabling the study of predictable tumor patterns from postnatal day 50. In this study, we characterized metabolic alterations during tumorigenesis (postnatal day 8 to day 50) and tumor growth (day 50 to day 65) in this model and explored the impact of PPARγ antagonism on human granulosa cell tumor proliferation. The tumor exhibited significant lipid accumulation, with PPARγ and the proliferation marker Ki67 co-localizing at postnatal day 65. Transcriptome analysis demonstrates that pathways for lipid metabolism and mitochondrial oxidation are promoted during tumorigenesis and tumor growth, respectively. Overlappingly upregulated genes during tumorigenesis and tumor growth are associated with lipid metabolism pathways. Correspondingly, mouse granulosa cell tumor shows overexpression of peroxisome proliferator-activated receptor gamma and DGAT2 proteins at postnatal day 65. Furthermore, GW9662 reduces the proliferation of KGN human granulosa cell tumor cells and decreases the phosphorylation of AKT and SMAD3. Our findings identify metabolic abnormalities in ooPIK3CA* granulosa cell tumor model and suggest peroxisome proliferator-activated receptor gamma as a potential driver for primary granulosa cell tumor growth.

Uncovering Tumor-Promoting Roles of Activin A in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with high incidence rates of metastasis and cachexia. High circulating activin A, a homodimer of inhibin ßA subunits that are encoded by INHBA gene, predicts poor survival among PDAC patients. However, it still raises the question of whether activin A suppression renders favorable PDAC outcomes. Here, the authors demonstrate that activin A is abundantly detected in tumor and stromal cells on PDAC tissue microarray and mouse PDAC sections. In orthotopic male mice, activin A suppression, which is acquired by tumor-targeted Inhba siRNA using cholesterol-modified polymeric nanoparticles, retards tumor growth/metastasis and cachexia and improves survival when compared to scramble siRNA-treated group. Histologically, activin A suppression coincides with decreased expression of proliferation marker Ki67 but increased accumulation of α-SMAhigh fibroblasts and cytotoxic T cells in the tumors. In vitro data demonstrate that activin A promotes KPC cell proliferation and induces the downregulation of α-SMA and upregulation of IL-6 in pancreatic stellate cells (PSC) in the SMAD3-dependent mechanism. Moreover, conditioned media from activin A-stimulated PSC promoted KPC cell growth. Collectively, our data provide a mechanistic basis for tumor-promoting roles of activin A and support therapeutic potentials of tumor activin A suppression for PDAC.

TAp63 determines the fate of oocytes against DNA damage.

Cyclophosphamide and doxorubicin lead to premature ovarian insufficiency as an off-target effect. However, their oocyte death pathway has been debated. Here, we clarified the precise mechanism of ovarian depletion induced by cyclophosphamide and doxorubicin. Dormant oocytes instead of activated oocytes with high PI3K activity were more sensitive to cyclophosphamide. Checkpoint kinase 2 (CHK2) inhibitor rather than GNF2 protected oocytes from cyclophosphamide and doxorubicin, as cyclophosphamide up-regulated p-CHK2 and depleted primordial follicles in Abl1 knockout mice. Contrary to previous reports, TAp63 is pivotal in cyclophosphamide and doxorubicin-induced oocyte death. Oocyte-specific Trp63 knockout mice prevented primordial follicle loss and maintained reproductive function from cyclophosphamide and doxorubicin, indicated by undetectable levels of BAX and cPARP. Here, we demonstrated that TAp63 is fundamental in determining the signaling of oocyte death against DNA damage. This study establishes the role of TAp63 as a target molecule of adjuvant therapies to protect the ovarian reserve from different classes of chemotherapy.

Adipose Tissue Wasting as a Determinant of Pancreatic Cancer-Related Cachexia.

Pancreatic cancer (PC) is the third leading cause of cancer-related death in the US, and its 5-year survival rate is approximately 10%. The low survival rates largely stem from diagnostic delay and the presence of significant adipose tissue and muscle wasting, commonly referred to as cachexia. Cachexia is present in nearly 80% of PC patients and is a key cause of poor response to treatment and about 20% of death in PC patients. However, there are few clinical interventions proven to be effective against PC-related cachexia. Different cancer types feature distinct secretome profiles and functional characteristics which would lead to cachexia development differently. Therefore, here we discuss affected tissues and potential mechanisms leading to cachexia in PC. We postulate that the most affected tissue during the development of PC-related cachexia is adipose tissue, historically and still thought to be just an inert repository for excess energy in relation to cancer-related cachexia. Adipose tissue loss is considerably greater than muscle loss in quantity and shows a correlation with poor survival in PC patients. Moreover, we suggest that PC mediates adipose atrophy by accelerating adipocyte lipid turnover and fibroblast infiltration.

Oocyte CTR1 is not essential for cisplatin-induced oocyte death of primordial follicle.

Accumulated evidence indicates that cisplatin, a platinum-based alkylating agent, causes preferential DNA damage to oocytes of primordial follicles (PFs) in the ovary, suggesting oocyte-favored accumulation of cisplatin. Copper transporter 1 (CTR1; Slc31a1 ) is implicated in facilitating cisplatin uptake in cells. Here we found that oocytes of PFs had constitutively higher expression of CTR1 than other cell types in mouse ovary. However, oocyte-specific Slc31a1 knockout was not sufficient to prevent cisplatin-induced depletion of PFs in vitro . Our data indicate that CTR1 would not be the only route for cisplatin to be transported inside the oocytes of PFs in the ovary.

Conjugated linoleic acid improves meiotic spindle morphology and developmental competence of heat-stressed bovine oocyte.

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 µM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P < 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P < 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P < 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P > 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

Distribution and size of lipid droplets in oocytes recovered from young lamb and adult ovine ovaries.

This study evaluated the distribution and size of lipid droplets (LDs) in oocytes recovered from young and adult ovine ovaries. Collected oocytes were categorised on the basis of their major diameter (small (SO), 70-90 µm; medium (MO), >90-110 µm; large (LO), >110-130µm) and were stained with Nile red to detect LDs. In adult and young oocytes, a diffuse pattern distribution of LDs was dominant in all classes except adult LO and young SO and LO. Larger LDs (i.e. >3µm) were mostly present in young SO and LO, whereas smaller LDs (1-3µm) were detected in the other adult and young oocyte categories.