Zootoxins and Domestic Animals: A European View.

Zootoxins are produced by venomous and poisonous species and are an important cause of poisoning in companion animals and livestock in Europe. Little information about the incidence of zootoxin poisoning is available in Europe, with only a few case reports and review papers being published. This review presents the most important zootoxins produced by European venomous and poisonous animal species responsible for poisoning episodes in companion animals and livestock. The main zootoxin-producing animal species, components of the toxins/venoms and their clinical effects are presented. The most common zootoxicoses involve terrestrial zootoxins excreted by the common toad, the fire salamander, the pine processionary caterpillar, and vipers. The lack of a centralized reporting/poison control system in Europe makes the evaluation of the epidemiology of zootoxin-induced poisonings extremely difficult. Even if there are many anecdotal reports in the veterinary community about the exposure of domestic animals to terrestrial and marine zootoxins, the number of published papers regarding these toxicoses is low. Climate change and its consequences regarding species distribution and human-mediated transportation are responsible for the emerging nature of some intoxications in which zootoxins are involved. Although new venomous or poisonous animal species have emerged in regions where they were previously unreported, zootoxins produced by native species remain the main concern in Europe. The diversity of poisonous and venomous animal species and the emerging nature of certain poisonings warrant the continuous update to such knowledge by veterinary professionals and animal owners. This review offers an overview about zootoxin-related poisonings in domestic animals in Europe and also provides important information from a health perspective.

Enniatin B1: Emerging Mycotoxin and Emerging Issues.

Although over the last 10 years several studies have focused on the emerging mycotoxins known as enniatins (ENNs), there is still a lack of knowledge regarding their toxicological effects and the development of a correct risk assessment. This is especially true for enniatin B1 (ENN B1), considered the younger sister of the widely studied enniatin B (ENN B). ENN B1 has been found in several food commodities and, as with other mycotoxins, presents antibacterial and antifungal properties. On the other hand, ENN B1 has shown cytotoxic activity, impairment of the cell cycle, the induction of oxidative stress, and changes in mitochondrial membrane permeabilization, as well as negative genotoxic and estrogenic effects. Overall, considering the paucity of information available regarding ENN B1, further studies are necessary to perform a risk assessment. This review summarizes information on the biological characteristics and toxicological effects of ENN B1 as well as the future challenges that this mycotoxin could present.

Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells.

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

Insulin-like growth factor 1 and the hormonal mediation of sibling rivalry.

In altricial animals, young are completely dependent on parents for provisioning. The ability to outcompete siblings to receive parental provisioning has clear fitness benefits, and may be mediated by hormones that influence growth. We analyzed the effects of insulin-like growth factor 1 (IGF-1) on body size, growth, and sibling rivalry in eastern bluebirds (Sialia sialis). To determine whether IGF-1 is upregulated in response to the competitive environment, we manipulated brood sizes and examined the effect on IGF-1 levels, nestling body size, growth rate, and behavior. In a separate experiment, we injected nestlings with exogenous IGF-1 to study its impacts on body size, growth rate, and sibling competition. Brood size manipulation did not influence endogenous IGF-1 levels, but male nestlings with higher IGF-1 levels early in the nestling period tended to have greater mass gain than males with lower IGF-1 levels. Nestlings with higher IGF-1 levels also tended to be fed more frequently by parents. In the injection experiment, IGF-1 injected individuals tended to be heavier than vehicle injected young by the end of the nestling period, which suggests that IGF-1 can influence mass gain in bluebirds. IGF-1 has been proposed to be a mediator of life-history strategies and post-hatching behavior. Our results suggest that although bluebird nestlings do not adaptively elevate IGF-1 in response to the presence or number of siblings, IGF-1 may influence growth during the nestling period. These findings shed light on sibling competition, life history strategies, and the hormones that underlie them.

In Vitro Effects of Enniatin A on Steroidogenesis and Proliferation of Bovine Granulosa Cells.

The emerging Fusarium mycotoxins enniatins (ENNs) have been the focus of new research because of their well-documented existence in various cereal and grain products. Research findings indicate that reproductive disorders may be caused by exposure to Fusarium mycotoxins, but little work has evaluated ENNs on reproductive function. Therefore, to determine the effects of ENNA on the proliferation and steroidogenesis of granulosa cells (GC), experiments were conducted using bovine GC cultures. In vitro, ENNA (1−5 µM) inhibited (p < 0.05) hormone-induced GC progesterone and estradiol production. The inhibitory effect of ENNA on estradiol production was more pronounced in small- than large-follicle GC. In large-follicle GC, 0.3 µM ENNA had no effect (p > 0.10) whereas 1 and 3 µM ENNA inhibited GC proliferation. In small-follicle GC, ENNA (1−5 µM) dramatically decreased (p < 0.05) GC proliferation. Using cell number data, the IC50 of ENNA was estimated at 2 µM for both follicle sizes. We conclude that ENNA can directly inhibit ovarian function in cattle, decreasing the proliferation and steroid production of GC.

Hormone regulation of thrombospondin-1 mRNA in porcine granulosa cells in vitro.

Thrombospondin-1 (THBS1) is involved in the process of angiogenesis and is down-regulated by insulin-like growth factor 1 (IGF1) in porcine granulosa cells (GC), but what other hormones regulate GC THBS1 and its role in follicular growth is unclear. Thus, six experiments were conducted to determine the influence of other hormones on THBS1 gene expression in porcine GC, and to determine if THBS1 mRNA changes during follicular development. For Exp. 1-5, small (1-5 mm) follicles from ovaries of abattoir gilts were aspirated, GC collected and treated with FSH, IGF1, fibroblast growth factor 9 (FGF9), Sonic hedgehog (SHH), estradiol, cortisol, and/or prostaglandin E2 (PGE2). FSH, IGF1 and FGF9 each decreased (P < 0.05) THBS1 mRNA abundance. Alone, PGE2 increased (P < 0.05) THBS1 mRNA abundance. PGE2 significantly attenuated the FSH-induced inhibition of THBS1 mRNA expression. Estradiol, cortisol, and SHH had no effect on THBS1 mRNA abundance. In Exp. 6, small (1-3 mm), medium (4-6 mm) and large (7-14 mm) follicles were aspirated to measure abundance of THBS1 mRNA in GC which did not differ (P > 0.10) between small and medium-sized follicles but was threefold greater (P < 0.05) in large compared to small or medium follicles. We hypothesize that the inhibitory effects of FSH, IGF1 and FGF9 on the antiangiogenic gene THBS1 could contribute to promoting angiogenesis in the developing follicle, while stimulation of THBS1 mRNA by PGE2 may help reduce angiogenesis during the preovulatory period when PGE2 and THBS1 mRNA are at their greatest levels.

Effects of grape phenolics, myricetin and piceatannol, on bovine granulosa and theca cell proliferation and steroid production in vitro.

Myricetin (a flavonol) and piceatannol (a stilbenoid) are naturally occurring phenolic compounds in red wine with cardio-protective and anti-carcinogenic effects, but their potential reproductive effects have not been investigated. Thus, the present study was designed to determine if myricetin and piceatannol can directly affect ovarian function using bovine granulosa cells (GC) and theca cells (TC) as in vitro model systems to evaluate effects on cell proliferation and steroid production. In Experiment 1 and 2, myricetin and piceatannol at 30 µM blocked insulin-like growth factor 1 (IGF1)-induced progesterone production by GC without affecting GC numbers. In contrast, myricetin stimulated IGF1-induced estradiol production, whereas piceatannol at 30 µM inhibited IGF1-induced estradiol production by 90% in GC. In Experiment 3 and 4, TC androstenedione and progesterone production and TC proliferation was inhibited by myricetin and piceatannol at 30 µM. In Experiment 5, piceatannol (30 µM) reduced the Fusarium mycotoxin, beauvericin (6 µM)-induced inhibition on progesterone production and cell proliferation. Myricetin (30 µM) reduced the inhibitory effect of beauvericin on estradiol but not progesterone production or cell proliferation. In conclusion, the red wine phenols, myricetin and piceatannol, directly affected GC and TC steroidogenesis, and were able to reduce some of the inhibitory effects of beauvericin on GC function.

Effects of transforming growth factor β1 on steroidogenesis of feline granulosa cells cultured in vitro.

CONTEXT: Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. AIMS: To determine if transforming growth factor ß1 (TGFB1), activin, epidermal growth factor (EGF), follicle stimulating hormone (FSH), luteinizing hormone (LH), melatonin, and insulin-like growth factor 1 (IGF1) regulate granulosa cell steroidogenesis and proliferation in cats, three experiments were conducted in winter season. METHODS: Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48h after an initial 48h plating in 10% fetal calf serum. KEY RESULTS: Treatment with IGF1 and FSH increased (P <0.05) estradiol production by 2.3- and 1.33-fold, respectively. In contrast, TGFB1 blocked (P <0.05) IGF1-induced estradiol production and inhibited FSH-induced estradiol production by 60%. Combined with FSH or FSH plus IGF1, TGFB1 inhibited (P <0.05) cell proliferation, whereas TGFB1 increased progesterone production by 2.8-fold in the presence of FSH plus IGF1. EGF decreased (P <0.05) FSH plus IGF1-induced estradiol production by 89% but did not affect progesterone production or cell numbers. Activin did not affect (P >0.10) cell numbers or steroidogenesis in the presence of FSH plus IGF1. Melatonin and LH decreased (P <0.05) estradiol production 53% and 59%, respectively, without affecting progesterone production or cell proliferation. CONCLUSIONS: The present study has identified TGFB1 as a major regulator of feline ovarian function, in addition to EGF, IGF1, melatonin, LH and FSH. IMPLICATIONS: These studies will provide useful information for future development of fertility control in feline species.

Emerging mycotoxins and reproductive effects in animals: A short review.

Emerging Fusarium mycotoxins beauvericin (BEA), enniatins (ENNs), and moniliformin (MON) are gaining increasing interest due to their wide presence especially in cereals and grain-based products. In vitro and in vivo studies indicate that Fusarium mycotoxins can be implicated in reproductive disorders in animals. Of these mycotoxins, BEA may affect reproductive functions, impairing the development of oocytes in pigs and sheep. Studies show dramatic inhibitory effects of BEA and ENNA on bovine granulosa cell steroidogenesis. ENNs also inhibit boar sperm motility and cause detrimental effects on embryos in mice and pigs. Although little data are reported on reproductive effects of MON, in vitro studies show inhibitory effects of MON on Chinese hamster ovary cells. The present review aims to summarize the reproductive toxicological effects of emerging Fusarium mycotoxins BEA, ENNs, and MON on embryo development, ovarian function, and testicular function of animals. In vitro and in vivo toxicological data are reported although additional studies are needed for proper risk assessment.

A potential role of fibrillin-1 (FBN1) mRNA and asprosin in follicular development in water buffalo.

Fibrillin-1 (FBN1) functions as a structural protein in the ovary, while the role of its protein product asprosin remains unknown. Both proteins are encoded by the FBN1 gene and when it is cleaved at the C-terminal end, asprosin is produced. Asprosin is associated with various metabolic parameters and sex-related hormones in women. One goal of this research was to quantify FBN1 and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) mRNA in water buffalo granulosa cells and correlate them to aromatase (CYP19A1) gene expression. A second goal was to determine the effect of asprosin on follicular growth in vivo. In Exp. 1, ovaries were collected from a local slaughterhouse, follicular fluid and granulosa cells from small (<6 mm) and large (6-13 mm) follicles were aspirated, cellular RNA extracted for gene expression analysis, data analyzed using ANOVA, and Pearson correlation coefficients were calculated among FBN1, OR4M1, and CYP19A1 gene expression. In Exp. 2, an intra-follicular injection of asprosin (600 ng of asprosin/194 µL of PBS) or vehicle (200 µL of PBS; Controls) was given via the theca layer of the dominant follicle of synchronized cows (n = 5/group) 1 day after injection of PGF2α, follicle sizes were measured daily via transrectal ultrasonography for 3 days, a two-way repeated measures ANOVA was used to determine the effect of asprosin on growth rate of follicles from day 0-2, and Chi-square analysis for the percentage of cows ovulated 2 days following asprosin injections. In Exp. 1, FBN1 mRNA abundance was 1.9-fold greater in cells of follicular aspirates from small than large follicles (P < 0.05), but abundance of OR4M1 and CYP19A1 mRNA did not differ (P > 0.10) between the two sizes of follicles. Abundance of FBN1 mRNA was positively correlated with CYP19A1 (r = 0.55, P < 0.05) and OR4M1 mRNA (r = 0.50, P < 0.06) across follicle sizes. In Exp. 2, cows treated with asprosin revealed a greater follicle growth rate from day 0-2 (63.4% increase in diameter) than placebo cows (36.8% increase in diameter) post-injection, and more follicles from asprosin treatment vs. control group (100% vs. 20%; P < 0.05) ovulated within 2 days. These findings suggest that FBN1 may be developmentally regulated in follicular cells, and that asprosin may induce follicular growth in buffaloes, but further studies will be required to determine if asprosin directly regulates estradiol production during follicle development.

Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

Wingless-type mouse mammary tumor virus integration site regulation of bovine theca cells.

Ovarian paracrine mediation by components of the wingless-type mouse mammary tumor virus integration site ligands (WNT1 to 11) and their receptors, frizzled family members (FZD1 to 10), has been proposed. Secreted truncated forms of FZD proteins (e.g., secreted frizzled-related protein 4 [SFRP4]) block the action of WNT ligands. Dickkopf-1 (DKK1) is another WNT antagonist, and R-spondin-1 (RSPO1) is one of a group of four secreted proteins that enhance WNT/ß-catenin signaling. Our hypothesis was that granulosa cells signal theca cells (TCs) via SFRP4, DKK1, RSPO1, and WNT secretion to regulate TC differentiation and proliferation. Therefore, in vitro experiments were conducted to study the effects of WNT family member 3A (WNT3A), WNT5A, RSPO1, DKK1, insulin-like growth factor 1 (IGF1), bone morphogenetic protein 7 (BMP7), Indian hedgehog (IHH), and fibroblast growth factor 9 (FGF9) on bovine TC proliferation and steroidogenesis. TCs of large (8 to 20 mm) and small (3 to 6 mm) follicles were collected from bovine ovaries; TC monolayers were established in vitro and treated with various doses of recombinant human WNT3A, WNT5A, RSPO1, DKK1, IGF1, FGF9, BMP7, IHH, and/or ovine luteinizing hormone (LH) in serum-free medium for 48 h. In experiment 1, using LH-treated TC, IGF1, IHH, and WNT3A increased (P < 0.05) cell numbers and androstenedione production, whereas WNT3A and BMP7 inhibited (P < 0.05) progesterone production. In experiment 2, FGF9 blocked (P < 0.05) the WNT3A-induced increase in androstenedione production in LH plus IGF1-treated TC. In experiment 3, RSPO1 further increased (P < 0.05) LH plus IGF1-induced progesterone and androstenedione production. In experiment 4, SFRP4 and DKK1 alone had no significant effect on TC proliferation or progesterone production of large-follicle TC but both blocked the inhibitory effect of WNT5A on androstenedione production. In contrast, DKK1 alone inhibited (P < 0.05) small-follicle TC androstenedione production whereas SFRP4 was without effect. We conclude that the ovarian TC WNT system is functional in cattle, with WNT3A increasing proliferation and androstenedione production of TC.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro.

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.

Discovery of a possible role of asprosin in ovarian follicular function.

Asprosin is a novel fasting-induced protein encoded by fibrillin-1 (FBN1) gene, produced when FBN1 is cleaved by the enzyme furin, and is associated with insulin resistance and polycystic ovarian syndrome in humans. To characterize mRNA abundance of FBN1, FURIN, and the presumed asprosin receptor, olfactory receptor family 4 subfamily M member 1 (OR4M1) in granulosa (GC) and theca cells (TC), and identify hormones regulating FBN1 mRNA expression, GC and TC from small (1-5 mm; SM) and large (>8 mm; LG) follicles were collected from ovaries of heifers obtained at an abattoir and used for real-time PCR gene expression analysis or in vitro evaluation of hormone regulation and asprosin effects. SMTC had 151-fold greater (P < 0.05) FBN1 mRNA abundance than SMGC, and LGTC had 50-fold greater FBN1 mRNA than LGGC. In contrast, OR4M1 mRNA was 81-fold greater in SMGC than LGGC and did not differ from SMTC, but LGTC had 9-fold greater OR4M1 mRNA than LGGC. FURIN mRNA was 2.6-fold greater in SMTC than SMGC, but did not differ among follicular sizes. In cultured TC, leptin, insulin, LH, IGF1 and steroids did not affect FBN1 mRNA, but TGFB1 increased (P < 0.05) FBN1 mRNA by 2.2-fold; EGF and FGFs increased FBN1 mRNA by 1.3- to 1.5-fold. Asprosin enhanced LH-induced TC androstenedione production, reduced IGF1-induced TC proliferation, and had no effect on progesterone production. Developmental regulation of FBN1, FURIN and OR4M1 along with direct effects of asprosin on TC suggests that asprosin may be a novel regulator of ovarian follicular function.

Influence of N-acetylcysteine on steroidogenesis and gene expression in porcine placental trophoblast cells.

N-acetylcysteine (NAC) is a widely used anti-inflammatory agent and antioxidant in vivo and in vitro. As a nutritional supplement, NAC can improve production and reproductive performances in animals through enhancing placental function and regulating hormone production. Trophoblast proliferation and steroid hormone production are two major functions in the placenta. We hypothesized that the effects of NAC on placental function is due to its direct and indirect effects on gene expression in placental trophoblast cells (pTr). To evaluate this hypothesis, we investigated the effects of NAC on steroidogenesis, gene expression, and cell proliferation in porcine pTr in vitro. pTr were treated with NAC in serum-free medium for 24 h with different concentrations (0, 0.1 µM, 1.0 µM, 10.0 µM, 0.1 mM, 1.0 mM, and 10.0 mM). Low-dose NAC (1 µM) stimulated pTr proliferation and decreased progesterone production, while increasing estradiol production (P < 0.05). High-dose NAC (10 mM) suppressed cell proliferation (P < 0.05), but had no effect on steroidogenesis. Low-dose NAC increased CCDN1 and decreased CASP3 and CASP8 mRNA levels (P < 0.05), whereas high-dose NAC decreased CDK4 and CCDN1 and increased CASP3 mRNA levels (P < 0.05). NAC had no effect on the mRNA abundance of StAR and HSD3B. Low-dose NAC upregulated CYP19A1 mRNA expression, and high-dose NAC downregulated CYP11A1 mRNA abundance (P < 0.05). Only low-dose NAC increased NOS3 mRNA abundance and tetrahydrobiopterin reduction (BH4/BH2 ratio). We conclude that NAC may act directly and indirectly on pTr with a dose-dependent manner and may regulate placental function by affecting pTr differentiation via regulating pTr steroid synthesis, cell proliferation, and apoptosis in sows.

Effects of N-carbamylglutamate on steroidogenesis and relative abundances of mRNA transcripts in pig placental trophoblasts.

Supplementation of N-carbamylglutamate (NCG) improves gestation outcomes, with increased piglet within-litter uniformity of birth weight and reduced peripheral steroid concentrations in pregnant sows and ewes. It was hypothesized that the effect of NCG on placental function results from direct effects on the placental trophoblasts. There, therefore, was investigation of the effects of NCG on pig placental trophoblast (pTr) steroidogenesis, mRNA transcript abundance, and cell proliferation in vitro. The pTr were treated with NCG in serum-free medium for 24-48 h. Treatment with NCG inhibited pTr progesterone, androstenedione, testosterone (all P <  0.01), and estradiol (P <  0.05) production, whereas it promoted (P <  0.05) pTr proliferation. Treatment with NCG suppressed (P <  0.05) the relative abundances of CYP11A1, CYP19A1, and CASP3 and increased abundances of CCDN1 (P <  0.01) and CDK4 (P <  0.05) mRNA transcripts in pTr, whereas NCG treatment had no effect (P >  0.10) on relative abundances of StAR, HSD17B4, or HSD3B mRNA transcripts. Treatments with NCG can increase pTr cell numbers of sows through upregulating CCND1 and CDK4 and suppressing CASP3 mRNA transcript abundances, while modulating steroidogenesis through effects on CYP11A1 and CYP19A1 mRNA transcript abundances. It is concluded that NCG may have a direct action on pTr and may regulate placental function by suppressing pTr differentiation as a consequence of lesser steroid synthesis while promoting pTr proliferation and inhibiting apoptosis in sows.

Developmental and hormonal regulation of ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 gene expression in ovarian granulosa and theca cells of cattle.

Ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) is a multi-domain nuclear protein that plays an important role in epigenetics and tumorigenesis, but its role in normal ovarian follicle development remains unknown. Thus, the present study evaluated if UHRF1 mRNA abundance in bovine follicular cells is developmentally and hormonally regulated, and if changes in UHRF1 are associated with changes in DNA methylation in follicular cells. Abundance of UHRF1 mRNA was greater in granulosa cells (GC) and theca cells (TC) from small (<6 mm) than large (≥8 mm) follicles and was greater in small-follicle GC than TC. In GC and TC, fibroblast growth factor 9 (FGF9) treatment increased (P < 0.05) UHRF1 expression by 2-fold. Also, luteinizing hormone (LH) and insulin-like growth factor 1 (IGF1) increased (P < 0.05) UHRF1 expression in TC by 2-fold, and forskolin (an adenylate cyclase inducer) alone or combined with IGF1 increased (P < 0.05) UHRF1 expression by 3-fold. An E2F transcription factor inhibitor (E2Fi) decreased (P < 0.05) UHRF1 expression by 44% in TC and by 99% in GC. Estradiol, progesterone, and dibutyryl-cAMP decreased (P < 0.05) UHRF1 mRNA abundance in GC. Treatment of GC with follicle-stimulating hormone (FSH) alone had no effect but when combined with IGF1 enhanced the UHRF1 mRNA abundance by 2.7-fold. Beauvericin (a mycotoxin) completely inhibited the FSH plus IGF1-induced UHRF1 expression in small-follicle GC. Treatments that increased UHRF1 mRNA (i.e., FGF9) in GC tended to decrease (by 63%; P < 0.10) global DNA methylation, and those that decreased UHRF1 mRNA (i.e., E2Fi) in GC tended to increase (by 2.4-fold; P < 0.10) global DNA methylation. Collectively, these results suggest that UHRF1 expression in both GC and TC is developmentally and hormonally regulated, and that UHRF1 may play a role in follicular growth and development as well as be involved in ovarian epigenetic processes.

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle.

The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P > 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

Regulation of the transcription factor E2F8 gene expression in bovine ovarian cells.

Overexpression of the transcription factor, E2F8, has been associated with ovarian cancer. Objectives of this study were to determine: 1) if E2F8 gene expression in granulosa cells (GC) and theca cells (TC) change with follicular development, and 2) if E2F8 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F8 mRNA abundance in GC and TC was greater (P < 0.05) in small than large follicles. FGF9 induced an increase (P < 0.05) in E2F8 mRNA abundance by 1.6- to 7-fold in large-follicle (8-20 mm) TC and GC as well as in small-follicle (1-5 mm) GC. Abundance of E2F8 mRNA in TC was increased (P < 0.05) with FGF2, FGF9 or VEGFA treatments alone in vitro, and concomitant treatment of VEGFA with FGF9 increased (P < 0.05) abundance of E2F8 mRNA above any of the singular treatments; BMP4, WNT3A and LH were without effect. IGF1 amplified the stimulatory effect of FGF9 on E2F8 mRNA abundance by 2.7-fold. Collectively, our studies show for the first time that follicular E2F8 is developmentally and hormonally regulated indicating that E2F8 may be involved in follicular development.

Hormonal regulation of vascular endothelial growth factor A (VEGFA) gene expression in granulosa and theca cells of cattle1.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis and is associated with increased vascularity in ovarian follicles of cattle. The objectives of this study were to investigate the developmental and hormonal regulation of VEGFA expression in ovarian granulosa and theca cells (TC) of cattle. Bovine ovaries were collected from a local slaughterhouse and granulosa cells (GC) and TC were collected from small (SM; 1 to 5 mm) and large (LG; 8 to 20 mm) follicles. Cells were collected fresh or cultured in serum-free medium and treated with various factors that regulate angiogenesis and follicular development. RNA was collected for analysis of VEGFA mRNA abundance via quantitative PCR. In SM-follicle GC (SMGC), prostaglandin E2 (PGE2) and FSH decreased (P < 0.05) VEGFA mRNA abundance by 30 to 46%, whereas in LG-follicle GC (LGGC), PGE2 and FSH were without effect (P > 0.10). In SMGC, dihydrotestosterone (DHT), sonic hedgehog (SHH), and growth differentiation factor-9 (GDF9) decreased (P < 0.05) VEGFA expression by 30 to 40%. Fibroblast growth factor-9 (FGF9) and estradiol (E2) were without effect (P > 0.10) on VEGFA mRNA in both SMGC and LGGC, whereas progesterone increased (P < 0.05) VEGFA mRNA in LGGC but had no effect in LGTC. Bone morphogenetic protein-4 (BMP4), LH, and FGF9 increased (P < 0.05) abundance of VEGFA mRNA by 1.5- to 1.9-fold in LGTC. Insulin-like growth factor-1 (IGF1) was without effect (P > 0.10) on VEGFA mRNA in both TC and GC. An E2F transcription factor inhibitor, HLM0064741 (E2Fi), dramatically (i.e., 8- to 13-fold) stimulated (P < 0.01) the expression of VEGFA mRNA expression in both SMGC and LGTC. Abundance of VEGFA mRNA was greater (P < 0.05) in LGGC and SMGC than in LGTC. Also, SMTC had greater (P < 0.05) abundance of VEGFA mRNA than LGTC. In conclusion, VEGFA mRNA abundance was greater in GC than TC, and VEGFA expression decreased in TC during follicle development. Some treatments either suppressed, stimulated, or had no effect on VEGFA expression depending on the cell type. The inhibition of E2F transcription factors had the greatest stimulatory effect of all treatments evaluated, and thus, E2Fs may play an important role in regulating angiogenesis during follicle growth in cattle.