Zinc (Zn) plays a crucial role in the maturation and fertilization of both female [1] and male [[2], [3], [4]] gametes. Recent research has highlighted the significant involvement of Zn in key mammalian reproductive processes. In oocytes, "zinc sparks" (rapid Zn releases into the external environment) are triggered by fertilization and are essential for cell cycle resumption [5]. Further research has demonstrated a link between Zn ion fluxes and sperm viability and fertility in mammals [6]. Moreover, a unique pattern of Zn ion redistribution (“Zn signature”) has recently been associated with in vitro sperm capacitation [6]. Beyond its role in gametes, Zn is involved in multiple reproductive processes [1,7,8] and cellular functions, such as gene transcription and enzyme activity [7,[9], [10], [11]]. Notably, Zn significantly affects the ability of the oocyte to develop into a blastocyst [12,13]. Moreover, Zn present during in vitro maturation reduces DNA damage and apoptosis in cumulus cells (CC), while increasing glutathione (GSH) levels and Cu/Zn-superoxide dismutase (SOD) activity [[12], [13], [14]]. Although the involvement of Zn in germinal and reproductive somatic cells (e.g., CC and granulosa cells) is recognized, little is known about the role of Zn in oviduct epithelial cells (OEC).

The OEC are crucial in mammalian reproduction, facilitating gamete transport, sperm capacitation, fertilization, and early embryonic development [[15], [16], [17]]. The OEC dynamically transform throughout the estrous cycle, undergoing morphological, biochemical, and physiological shifts to optimize the oviduct environment for successful reproduction. Steroid hormones, particularly estradiol (E2) and progesterone (P4), regulate these changes, influencing gene expression and protein abundance [18]. Given the essential role of bovine OEC (BOEC) in sperm maturation, fertilization, and early embryo development, and the fundamental importance of Zn for cellular function [11,19], understanding Zn homeostasis in BOEC is vital. This is particularly relevant since cellular Zn homeostasis is influenced by hormonal stimuli [[20], [21], [22]], as already shown in oocytes and CC [23]. Zn homeostasis is maintained by tightly controlled uptake, storage, distribution, and efflux, primarily regulated by Zn transporter (ZnT) and Zrt-, Irt-related protein (ZIP) transporters [21].

Our hypothesis proposed that E2, P4, and Zn, at physiological concentrations, play a role in regulating Zn homeostasis within BOEC. To explore this, we analyzed whether E2, P4, and Zn alter intracellular free Zn levels and the expression of specific Zn transporters. Short-term (24 h) in vitro BOEC cultures were utilized to preserve their morphological integrity and responsiveness to hormonal stimuli [24]. Then, intracellular Zn concentration was evaluated by FluoZin-3AM fluorescent staining, while the gene expression of transporters ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9 was evaluated through real-time polymerase-chain reaction (RT-PCR).