In recent years, vitamin D₃ has been revealed as an important regulator of reproductive processes in humans and livestock; however, its role in the female reproductive system of poultry is poorly known. The aim of this study was to examine vitamin D₃ receptor (VDR and PDIA3) and metabolic enzyme (1α-hydroxylase and 24-hydroxylase) mRNA transcript and protein abundances, and protein localization within the hen ovary, oviductal shell gland, pituitary, liver, and kidney. We demonstrated, for the first time, the patterns of the relative mRNA and protein abundances of examined molecules in the ovary, dependent on follicle development and the layer of follicle wall, as well as in other examined organs. Immunohistochemically, PDIA3, 1α-hydroxylase, and 24-hydroxylase are localized in follicular theca and granulosa layers, luminal epithelium and tubular glands of the shell gland, pituitary, liver, and kidney. These results indicate that reproductive tissues have both receptors, VDR, primarily involved in genomic action, and PDIA3, probably participating in the rapid, non-genomic effect of vitamin D₃. The finding of 1α-hydroxylase and 24-hydroxylase expression indicates that the reproductive system of chickens has the potential for vitamin D₃ synthesis and inactivation, and may suggest that locally produced vitamin D₃ can be considered as a significant factor in the orchestration of ovarian and shell gland function in hens. These results provide a new insight into the potential mechanisms of vitamin D₃ action and metabolism in the chicken ovary and oviduct.

The role of vitamin D₃ has been confirmed in female reproductive organs. This study aimed to examine vitamin D₃ metabolic enzymes, i.e., CYP27B1 and CYP24A1, mRNA transcript and protein abundance, and protein localization in the uterus of pigs on days 2–5, 10–12, 15–16 and 18–20 of the estrous cycle. Additionally, we determined 1,25(OH)₂D₃ concentration in uterine flushings and the effect of 1,25(OH)₂D₃ (10, 50 and 100 ng/mL) in vitro on CYP27B1 and CYP24A1 mRNA transcript abundance in endometrial and myometrial slices. In the endometrium, a greater CYP27B1 mRNA transcript abundance was noted on days 10–12 and 18–20 than on days 15–16, whereas encoded protein abundance was greater on days 18–20 when compared to days 15–16. Endometrial CYP24A1 mRNA transcript abundance was greater on days 18–20 than on days 10–12 and 15–16. In the myometrium, CYP27B1 mRNA transcript abundance was greater on days 18–20 than on days 2–5 and 15–16, while protein abundance was larger in slices collected on days 18–20 than on days 15–16. Neither CYP24A1 mRNA transcript nor encoded protein abundance were detected in the myometrium. The highest 1,25(OH)₂D₃ concentration in uterine flushings was observed on days 18–20. Furthermore, the 1,25(OH)₂D₃ increased the abundance of the CYP24A1 mRNA transcript in endometrial slices. Overall, our results suggest that porcine uterus is an extra-renal site of vitamin D₃ metabolism. Both the endometrium and the myometrium possess the ability to synthesize vitamin D₃, while only the endometrium contributes to its catabolism.

The objective of the study was to investigate the effect of dietary supplementation with nettle or fenugreek on folliculogenesis and steroidogenesis in the juvenile rabbit ovary. To gain insight into the mechanism of action of these herbs, we examined follicle formation, ovarian cell proliferation and apoptosis, steroidogenic enzyme abundance and steroid concentrations in ovarian tissue and plasma. Animals were fed with control, 1% nettle- or 1% fenugreek-supplemented pellets from 5 to 12 weeks of age (n = 10 per each group), when animals were slaughtered for ovary and blood collection. The addition of nettle decreased the numbers of primordial (P = 0.015) and early antral (P = 0.02) follicles and increased the number of primary (P = 0.04) ones when compared with the control group. Following fenugreek supplementation, the numbers of primary (P = 0.008) and antral (P = 0.027) follicles were greater, while the number of early antral (P = 0.003) follicles was lower in comparison with the control group. Nettle revealed apoptotic activity through activation of caspases 9 (P = 0.047), 8 (P = 0.022) and 3 (P = 0.004), whereas fenugreek increased (P = 0.042) follicular cell proliferation marked by PCNA protein abundance. Furthermore, only fenugreek targeted steroidogenic enzymes, decreasing CYP17A1 (P = 0.043) and increasing CYP19A1 (P = 0.048) protein abundances that resulted in enhanced estradiol biosynthesis and its elevated (P = 0.006) plasma concentration. In conclusion, both herbs affected follicle development in the rabbit ovary in a stage specific manner. Additionally, fenugreek altered ovarian steroidogenesis in a way that might affect sexual maturation in rabbits.

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder with disrupted uterus structure and function. A positive effect of vitamin D₃ (VD₃) in female reproduction was observed. Chemerin (RARRES2) and adiponectin (ADIPOQ) are the main adipokines whose levels are altered in PCOS patients. Therefore, the aim of this study was to investigate the impact of VD₃ supplementation on RARRES2 and ADIPOQ levels in the uterus of PCOS rats. Methods: We analyzed the plasma levels and uterine transcript and protein expression of RARRES2 and ADIPOQ and their receptors (CCRL2, CMKLR1, GPR1, and ADIPOR1 and ADIPOR2, respectively) in rats with letrozole-induced PCOS. Results: In control animals, VD₃ did not change plasma levels of both adipokines, while in PCOS rats supplemented with VD₃, they returned to control levels. The expression of RARRES2 and all investigated receptors increased in the uterus of VD₃-treated rats; however, the levels of Rarres2 and Gpr1 genes remained unchanged. VD₃ supplementation decreased RARRES2, CMKLR1, and GPR1 but increased CCRL2 level to the control value. In the uterus of VD₃-treated rats, the transcript and protein levels of ADIPOQ and both receptors ADIPOR1 increased. At the same time, VD₃ supplementation induced an increase in Adipoq, Adipor1, and Adipor2 gene expression and restored protein levels to control level values. Conclusions: our findings indicate a new mechanism of VD₃ action in the uterine physiology of PCOS rats.

The study aimed to investigate whether vitamin D₃ and insulin alone or in co-treatment can influence characteristics and protein cargo of the porcine follicular fluid (FF)-derived extracellular vesicles (EVs). To this end, medium antral follicles were cultured in vitro alone (C; control) or with 1α,25(OH)₂D₃ (VD; 100 ng/mL) and insulin (I; 10 ng/mL) separately or in combination (VD+I). The properties of the FF-EVs were assessed by transmission electron microscopy, nanoparticle tracking analysis, electroforetic light scattering, and flow cytometry, while the global proteomic analysis was conducted by liquid chromatography-tandem mass spectrometry coupled with the TMT-isobaric mass tag labeling. In all groups, particles represented a typical cup-shaped morphology, ranged between 50 and 450 nm in diameter, and expressed marker proteins, such as CD63 and CD81. The I treatment decreased the concentration and average size of the FF-EVs, while VD reversed only the effect on the particle concentration. Proteomic analysis revealed 48 differentially abundant proteins (DAPs) between examined groups, whilst greater amount of DAPs was identified following VD and I treatment alone than in co-treatment. Functional analysis showed that VD alone or in combination with I decreased predominantly the abundance of ribosomal proteins. In the I group, proteins involved in oxidative stress were down-regulated. We also found that the FF-EVs are carriers of adiponectin, which was up-regulated in the VD+I group. To sum up, VD and I seem to be novel modulators of the porcine FF-EVs characteristics and protein cargo, and thereby could modify ovarian follicle function via the EV-mediated pathway.