Progress 05/01/24 to 04/30/25
Outputs
Target Audience:Findings were presented to scientist and industryprofessionals at intramurall, regional and national meetings and to prospective undergraduate studetns in the Departmetn of Animal Sciences at Penn State University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate trainees are now working partly or wholly on this project. One trainee has completed her dissertation, successfully defended her thesis, and has one manuscript under review and another in preparation. Both trainees have presented their work at intramural, regional, and international meetings. Graduate students also mentor undergraduates and technicians in procedures such as the isolation of mural granulosa cells and biochemical assays, including ELISA and Western blots. Students had the opportunity to collect ovaries from a local packing plant and perform follicle aspirations and cell culture. How have the results been disseminated to communities of interest?Results were presented at intramural, regional and national meetings. Several abstracts have been published and a thesis has been accepted for publication by the Graduate School at Penn State. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will focus on optimizing CRISPR/Cas9-mediated gene editing of ZIP9 in bovine granulosa cells. Our current editing efficiency is approximately 35%, and we aim to improve this through refinements in guide RNA design and transfection protocols. Achieving higher efficiency will be critical for downstream functional studies of ZIP9. We also plan to continue mechanistic experiments using ZIP9 agonists, pep-1 (a tetrapeptide previously shown to activate ZIP9) and T4-BSA (testosterone conjugated to bovine serum albumin). These studies will examine the effects of ZIP9 activation on key aspects of granulosa cell function, including steroidogenesis, zinc flux, and extracellular matrix (ECM) deposition. Steroidogenic outcomes will be assessed through quantification of progesterone and estradiol production via ELISA, while changes in zinc homeostasis will be monitored using zinc-sensitive fluorophores. ECM-related gene expression and protein deposition (e.g., collagen) will be evaluated by qPCR and Western blotting. Lastly, we will initiate untargeted metabolomic profiling of follicular fluid samples obtained from cattle with high and low androstenedione levels. This effort aims to identify metabolic signatures associated with hyperandrogenism and to uncover potential biomarkers or pathways involved in the altered follicular environment. Key metabolites will be used as biomarkers for in vitro studies using pep-1 and T4-BSA. Metabolomic analysis will be conducted using high-resolution mass spectrometry, and results will be integrated with hormone data to better understand the metabolic consequences of androgen excess in the ovarian follicle. These efforts will significantly advance our understanding of ZIP9 function and the broader impact of androgen signaling on ovarian physiology and pathology.
Impacts
What was accomplished under these goals?
During the past reporting period, we continued to expand our observations on the role of zinc ions in granulosa cell function. Previously, we showed that bovine granulosa cells incubated with increasing levels of a zinc chelator, TPEN (2.5, 3.5, 5, and 10 µM), for 24 hours exhibited a modest decrease in cell viability at 3.5 µM TPEN (p = 0.003), while a severe decrease occurred at 5 and 10 µM TPEN (p = 1.4 × 10-7and 4.6 × 10-8, respectively). We now show an upregulation of Caspase-3/7 activity at 10 µM TPEN. Furthermore, we demonstrate that the Hippo pathway participates in the TPEN-induced reduction in viability. The Hippo pathway is a known regulator of granulosa cell fate decisions--whether to proliferate, remain quiescent, or undergo cell death. At 10 µM TPEN, there was an upregulation of phosphorylation of the Hippo pathway target YAP at Ser127, indicating activation by TPEN. To determine whether the Hippo pathway is causally linked to the observed TPEN-mediated apoptosis, a potent LATS1/2 inhibitor (10 µM TRULI) was used in combination with TPEN treatments. LATS1/2 is the kinase that phosphorylates YAP at Ser127. Inhibition of this kinase partially reversed the TPEN-induced loss of cell viability, indicating that zinc is involved in LATS1/2 activation. Surprisingly, TPEN treatment for 6 hours led to a 6-9-fold increase in the abundance of the YAP-regulated genes CCN1 and CCN2. This was unexpected but clearly shows that TPEN is a potent regulator of Hippo signaling in granulosa cells. It is well known that androgens and androgen signaling are necessary for proper ovarian function, but excessive androgen production from the ovary is associated with ovarian dysfunction. While the mechanisms behind this androgen-induced dysfunction are still being elucidated, a novel membrane androgen receptor and zinc transporter not yet characterized in the bovine ovary, Zrt- and Irt-like protein 9 (ZIP9) which is an intriguing candidate for studying both physiological and pathophysiological androgen action in the bovine ovary. We now show that ZIP9 is expressed in bovine granulosa cells on the cell surface, and intriguingly, on the perinuclear membrane and the spindle of dividing granulosa cells. ZIP9 expression was not regulated by zinc availability, as its expression did not change when zinc was chelated by TPEN. Treatment with two ZIP9 agonists, testosterone conjugated to bovine serum albumin (T4-BSA) and the tetrapeptide IAPG (pep-1), previously shown to activate ZIP9 in other cells resulted in a dose-dependent decrease in absorbance as measured by an MTT assay. However, pep-1 treatment up to 100 µM did not decrease cell number as measured by direct counting, nor did it induce increased caspase-3/7 activity. This discrepancy between the MTT results and cell number was not due to dramatic changes in mitochondrial metabolism. Pep-1 did not cause a decrease in either mitochondrial or non-mitochondrial respiration, as measured by a sensitive Seahorse mitochondrial metabolism assay. Brightfield imaging of bovine mGCs after 48 hours of pep-1 treatment, followed by either a 1- or 4-hour incubation with MTT, revealed an increase in formazan crystal production in response to pep-1 treatment. This likely caused cell lysis, preventing further MTT reduction. While pep-1 and T4-BSA do not reduce mitochondrial metabolism per se, they do affect mitochondrial processing of formazan in the MTT assay, which could be related to changes in cholesterol metabolism. This hypothesis is currently being tested. In summary, activation of ZIP9 with specific agonists results in altered MTT assay kinetics, which may be associated with disrupted cholesterol metabolism and could contribute to defects in ovarian function. To characterize the in vivo prevalence of hyperandrogenism in the ovaries used for granulosa cell isolation, we measured follicular fluid androstenedione concentrations in 42 random samples from ovaries collected at the abattoir. We found a wide range (0.81 to 298 ng/mL) of androstenedione concentrations. This confirms the reported prevalence of 20-25% of cattle with high androstenedione levels (>20 ng/mL) in our general population. These samples will be used for metabolomic analysis.
Publications
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2025
Citation:
Carothers, A. (2025). Zinc-Dependent Signaling and ZIP9 Function in Mammalian Mural Granulosa Cells. Accepted.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Ibadin NO and Diaz FJ. Differential Effects of Potassium Channel Modulators on Mouse Sperm Motility and Function. Society for the Study of Reproduction, July 29-August 5, 2025, Washington D.C.
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Findings were presented to scientist and professionals at international scientific meetings and to graduate students and faculty at meetings and seminars at Penn State University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training Activities:Traineeworked with mentor nd other faculty to develop cell culture protocols for isolation and cultur eof bovine granulosa cells. In vitro assays for granulo cell funciton including proliferation and gene expression were developed. Professional Activities:Trainee presented work at an international coference. Tainee also was a teaching assistant for 2 courses in physiology and metntored an undegraduate student researcher. How have the results been disseminated to communities of interest?Findings werepresented at international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?1. The effect of ablation of the cell surface protein ZIP9 will be acomplished using AAV viruses or siRNA transfection. 2. Effect of androgen on zinc, G-protein and hippo pathway will be determined.
Impacts
What was accomplished under these goals?
High levels of follicular androgen are associated with reproductive dysfunction. However, the intraovarian consequences of high follicular androgen exposure are not known.Zip9 (Slc39a9) is a zinc transporter that moves zinc accross membranes and is also an androgen receptor. Zinc deficiency causes ovulatory dysfunction including defects in oocyte maturation andcumulus expansion in rodens, but the effect of zinc defficiency in bovine granulosa cells is not clear. The hypothesisthat zinc insufficiency would decrease cell viability in bovine granulosa cells was tested. Bovine granulosa cells collected from slaugtherhouse ovaries were incubated with differing levels of the zinc chelator (TPEN) (2.5, 3.5, 5, and 10 µM) for 24 hours. Cell viability was measured with an MTT assay. A moderate decrease in cell viability was seen at 3.5 µM TPEN, while a severe decrease was seen at 5 and 10 µM TPEN (n = 4; p<0.05). This decrease in cell viability was rescued with the addition of 10 and 20 µM ZnSO4, confirming no off-target effects of TPEN (n=4; p<0.05). TPEN has a high affinity for zinc, but lower affinity for copper, iron, and manganese. Chelators specific for these other ions(iron chelator = 10 µM 2,2'-Dipyridyl; copper chelator = 10 µM Bathocuproinedisulfonic acid disodium salt; manganese chelator = 10 µM Sodium-4-aminosalicyate dihydrate) were used to exclude off taget effecs and confirm the specificity of zinc in acutely regulating bovine granulosa cell viability. None of the other chelators caused adecrease in cell viability compared to the vehicle control, confirming zinc insufficiency is responsible for the decrease in cell viability in response to TPEN (n=4; p>0.05). These results demonstrate that zinc is an essential ion for bovine mural granulosa cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
22023
Citation:
Carothers A and Diaz FJ. Hormonal Regulation and Zinc Influx Activity of Zrt- and Irt-like Protein 9 (ZIP9) in the Mammalian Ovary. Society for the Study of Reproduction, July 11-14, 2023, Ottawa, Canada.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Carothers A and Diaz FJ. Role of Zinc Insufficiency in Bovine Granulosa Cell Viability. Society for the Study of Reproduction, July 15-19, 2024, Dublin, Ireland.
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