Functional and Morphological Characterization of Small and Large Steroidogenic Luteal Cells From Domestic Cats Before and During Culture

Abstract

The current study aimed to isolate, culture and characterize small (SLC) and large (LLC) steroidogenic cells from the corpora lutea (CL) of non-pregnant domestic cats. Isolation of feline SLC was based on an enzymatic digestion of luteal tissue, whereas LLC were obtained by mechanical disruption of CL. To assess function of both cell types, progesterone secretion and mRNA expression of selected genes involved in steroid and prostaglandin synthesis were measured, as well as relative transcript abundance of hormone receptors and anti-oxidative enzymes, before and during culture. The cells were cultured for 3 or 5 days without gonadotropins. Isolated feline SLC and LLC had different sizes (12 ± 3 μm vs. 34 ± 5 μm, respectively), morphologies (amount of lipid droplets) and behaved differently in culture. SLC attached and proliferated or spread quickly, but lost their steroidogenic function during culture (significant decrease in progesterone secretion and expression of steroidogenic genes). The expression of receptors for gonadotropins and prolactin also decreased. Prostaglandin synthase (PTGS2) decreased steadily over time, whereas mRNA expression of PGE2 synthase (PGES) increased. The gene expression of anti-oxidative enzyme glutathione peroxidase 4 (GPX4), also increased during culture, but not of superoxide dismutase 1 (SOD1). In comparison to SLC, LLC did not attach to culture plates, secreted more progesterone per inoculated cells and maintained steroidogenic function during culture. Expression of prostaglandin synthases (PTGS2 and PGES) was almost non-detectable. The gene expression of hormone receptors for prostaglandin F2 alpha (PTGFR), gonadotropins (LHCHR and FSHR), and prolactin (PRLR), as well as of anti-oxidative enzymes (GPX4, SOD1), increased over time. To conclude, we successfully isolated and cultured different types of feline steroidogenic luteal cells and comprehensively characterized both isolated cell types. This knowledge can be used to better understand the CL lifecycle in felines more broadly, and the established cell cultures will provide a foundation for future studies on luteolytic and luteotrophic factors in the domestic cat, and for comparison with other feline species, particularly lynx.

Luteinizing Hormone Effect on Luteal Cells Is Dependent on the Corpus Luteum Stage in Felids

“…Enzymatic isolation of small luteal cells from the domestic cat has been described previously [ 20 ] and was used without modifications in this study. Briefly, CLs were chopped on a petri dish with culture medium (HAM’s F12 and MEM Eagle medium 1:1 (v:v) supplemented with 5% of fetal bovine serum (FBS) and 0.055 mg/mL gentamicin).…”

“…Identification of steroidogenic luteal cells was performed by determination of enzyme activity of 3β-hydroxysteroid dehydrogenase (HSD3B) in freshly isolated and cultured cells [ 20 ]. The assay was performed on day two of culture.…”

“…On the following day, cells were washed twice in DPBS and the staining intensity within cells was documented by microphotography. For control, activity of 3β-hydroxysteroid dehydrogenase was blocked by 2 mM trilostane as described before [ 20 ].…”

“…The experiments we performed on CL from the formation, development/maintenance and early and late regression stages. The enzymatic isolation method and culture conditions previously described for domestic cats were applied [ 20 ]. The presented studies were conducted to contribute to knowledge about CL of felids.…”

The antioxidative enzyme SOD2 is important for physiological persistence of corpora lutea in lynxes

“…Quantifications were performed using label free quantification (LFQ) of the MaxQuant software (minimum ratio count 3). : GGC AGA AGC ACA GCC TCC SOD2-rv: TTC TGC TCA GTG TAA TGA TGT SOD2-q-fw: CAC ATC AAC GCC CAG ATC SOD2-q-rv: CAC CCT TAG GGC TCA GGT T 53 66 .…”