NR4A1-mediated regulation of lipid droplets in progesterone synthesis in goat luteal cells†.

Nuclear receptor NR4A1 is a key factor in glycolipid metabolism and steroidogenesis, while lipid droplets serve as crucial dynamic organelles for lipid metabolism in luteal cells. To investigate the effects of NR4A1 on lipid droplet metabolism and progesterone (P4) synthesis in goat corpus luteum in vitro, luteal cells from the middle-cyclic corpus luteum were isolated and treated with Cytosporone B (CSNB, an agonist) or siRNA of NR4A1. Results showed that both low (1 µM) and high (50 µM) concentrations of CSNB promoted lipid droplet accumulation, while NR4A1 knockdown reduced lipid droplet content. CSNB increased while siNR4A1 decreased total cholesterol content; however, CSNB and siNR4A1 did not change triglyceride content. CSNB increased the expression of perilipins at mRNA and protein levels, also increased LDLR, SCARB1, SREBFs, and HMGCR mRNA abundance. Treatment with siNR4A1 revealed opposite results of CSNB, except for HMCGR and SREBF2. For steroidogenesis, 1 µM CSNB increased, but 50 µM CSNB inhibited P4 synthesis, NR4A1 knockdown also reduced the P4 level. Further analysis demonstrated that 1 µM CSNB increased the protein levels of StAR, HSD3B, and P-HSL, while 50 µM CSNB decreased StAR, HSD3B, and CYP11A1 protein levels. Moreover, 50 µM CSNB impaired active mitochondria, reduced the BCL2, and increased DRP1, Caspase 3, and cleaved-Caspase 3 protein levels. siNR4A1 consistently downregulated the P-HSL/HSL ratio and the steroidogenic protein levels. In conclusion, NR4A1-mediated lipid droplets are involved in the regulation of progesterone synthesis in goat luteal cells.

RGMb expression in goat uterine tissues: possible role of RGMb in the proliferation and apoptosis of endometrial epithelial cells.

Context Bone morphogenetic proteins (BMPs) play an important role in the uteri. Repulsive guidance molecule b (RGMb; a.k.a. Dragon) has been confirmed as the coreceptor of BMPs to function through drosophila mothers against decapentaplegic protein (Smads) and mitogen-activated protein kinases (MAPK) pathways. We hypothesise that RGMb regulates the uterine function through the Smads and MAPK pathways. Aims This study aimed to investigate the expression of RGMb in goat uteri and the potential role of RGMb in the endometrial epithelial cells (EECs). Methods The localisation of RGMb in goat uterine tissues was detected by immunohistochemistry (IHC), EECs were isolated and transfected with siRNA to investigate the role of RGMb in proliferation, and apoptosis. The expression levels of Smads and MAPK members was measured by western blot (WB) and real-time PCR (RT-PCR). Key results IHC showed that RGMb was localised in goat endometrial luminal cells, glandular epithelial cells, and circular muscle fibres, but not in stromal cells. RT-PCR results showed that treatment with RGMb siRNA suppressed the expressions of proliferation-related genes cyclin D1 (CCND1 , P =0.0291), cyclin-dependent kinase 2 (CDK2 P =0.0107), and proliferating cell nuclear antigen (PCNA, P =0.0508), leading to the reduced viability of EECs (P =0.0010). WB results showed that the expression ratio of cleaved-caspase 3/caspase 3 (P =0.0013) was markedly increased after RGMb siRNA transfection. Likewise, the level of phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2, P =0.0068) and p-Smad1/5/8 (P =0.0011) decreased significantly, while there were no appreciable differences in the level of p-P38 MAPK expression (P >0.05). Conclusions RGMb might participate in the regulation of cell proliferation and apoptosis through Smads and ERK signalling pathways in goat EECs. Implications RGMb is involved in regulating the proliferation and apoptosis in goat endometrial epithelial cells.

Quercetin alleviated H2 O2 -induced apoptosis and steroidogenic impairment in goat luteinized granulosa cells.

Quercetin (Que) is a natural flavonoid in most plants. Luteinized granulosa cell (LGC) culture is necessary for the study of follicle growth/differentiation. In the present study, we analyzed the role of Que in steroid production and apoptosis in hydrogen peroxide (H2 O2 )-treated goat LGCs. The results showed that treatment with H2 O2 induced apoptosis in goat LGCs, and treatment with Que decreased LGC apoptosis induced by H2 O2 (P < .05), accompanied with the different expressions of BAX, BCL-2, Caspase 3, and Cleaved caspase 3. Meanwhile, the messenger RNA expressions of nuclear factor erythroid 2 like 2 (Nrf2) and its downstream genes were upregulated with H2 O2 +Que treatment, accompanied by the increased cellular viability (P < .05). Furthermore, Que alleviated H2 O2 -induced reduction in the secretion of progesterone (P4 ) (P < .05); however, it had no effect on the secretion of estrogen (E2 ). Simultaneously, the expressions of StAR and P450scc were increased when treated with Que +H2 O2 , compared with the group treated with only H2 O2 (P < .05). In conclusion, it is observed that Que could alleviate the H2 O2 -induced apoptosis and steroidogenic impairment in goat LGCs, which might be mediated by the Nrf2 pathway.

Roles of poly (ADP-ribose) polymerase 1 activation and cleavage in induction of multi-oocyte ovarian follicles in the mouse by 3-nitropropionic acid.

3-nitropropionic acid (3-NPA) is known to be a mitochondrial toxin produced by plants and fungi, which may produce DNA damage in cells. However, studies of its reproductive toxicology are lacking. We know that poly(ADP-ribose) polymerase (PARP) plays an important role in a large variety of physiological processes and is involved in DNA repair pathways. The present study was therefore aimed at exploring the involvement of PARP-1 activation and cleavage after 3-NPA stimulation in female mice. We observed an increased number of atretic follicles and multi-oocyte follicles (MOFs) after treatment with 3-NPA and serum concentrations of 17ß-oestradiol and progesterone were significantly reduced. Our results provide evidence that PARP-1 cleavage and activational signals are involved in pathological ovarian processes stimulated by 3-NPA. In addition, total superoxide dismutase, glutathione peroxidase and catalase activities were significantly increased, whereas succinate dehydrogenase was decreased in a dose-dependent manner. Results from our in vitro study similarly indicated that 3-NPA inhibited the proliferation of mouse granulosa cells and increased apoptosis in a dose-dependent manner. In summary, 3-NPA induces granulosa cell apoptosis, follicle atresia and MOFs in the ovaries of female mice and causes oxidative stress so as to disrupt endogenous hormonal systems, possibly acting through PARP-1 signalling.

Contemporaneous effects of diabetes mellitus and hypothyroidism on spermatogenesis and immunolocalization of Claudin-11 inside the seminiferous tubules of mice.

BACKGROUND: Diabetes and hypothyroidism produce adverse effects on body weight and sexual maturity by inhibiting body growth and metabolism. The occurrence of diabetes is always accompanied with thyroid dysfunction. Thus, it is important to take hypo- or hyper-thyroidism into consideration when exploring the adverse effects caused by diabetes. Previous reports have found hypothyroidism inhibits testicular growth by delaying Sertoli cell differentiation and proliferation. Hence, by establishing a mouse model of diabetes combined with hypothyroidism, we provided evidence that poly glandular autoimmune syndrome affected testicular development and spermatogenesis. RESULTS: we mimicked polyglandular deficiency syndrome in both immature and prepubertal mice by induction of diabetes and hypothyroidism, which caused decreases in serum concentrations of testosterone and insulin like growth factor 1 (IGF-1). Such reduction of growth factor resulted in inhibition of testicular and epididymal development. Moreover, expressions of Claudin-11 were observed between Sertoli cells and disrupted in the testes of syndrome group mice. We also found reduced sperm count and motility in prepubertal mice. CONCLUSIONS: This mimicry of the diabetes and thyroid dysfunction, will be helpful to better understand the reasons for male infertility in diabetic-cum-hypothyroid patients.

Temporal regulation of extracellular signal-regulated kinase 1/2 phosphorylation, heat shock protein 70 and activating transcription factor 3 during prostaglandin F-induced luteal regression in pseudopregnant rats following heat stress.

The aim of the present study was to investigate the effects of heat stress on heat shock protein (HSP) 70 expression and mitogen-activated protein kinase (MAPK) and protein kinase (PK) B signalling during prostaglandin F (PGF)-induced luteal regression. During pseudopregnancy, rats were exposed to heat stress (HS, 40°C, 2h) for 7 days and treated with PGF or physiological saline on Day 7; serum and ovaries were collected 0, 1, 2, 8 or 24h after PGF treatment. The early inhibitory effect of PGF on progesterone was reduced in HS rats. HSP70 expression in response to PGF was significantly enhanced in HS rats. PGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was significantly greater in the HS group; however, HS rats exhibited elevated basal levels of phosphorylation of p38 MAPK, but not ERK1/2. PGF treatment increased expression of activating transcription factor (ATF) 3 at 2h, which was inhibited by heat stress. Evaluating PKB signalling revealed that phosphorylation of p-Akt (Thr308 and Ser473) was reduced at 8 and 24h after PGF treatment in both non-heat stress (NHS) and HS groups, but there were no significant differences between the HS and NHS groups at any of the time points. In conclusion, the present study provides further evidence that heat stress may enhance HSP70 and affect ERK1/2 and ATF3 expression, but not Akt activation, during PGF-induced luteal regression in pseudopregnant rats.

Effects of Daily Exposure to Saccharin and Sucrose on Testicular Biologic Functions in Mice.

Saccharin sodium consumption is considered safe and beneficial, owing to its very intense sweetness without any associated calories, but supporting scientific data remain sparse and controversial. Herein, we demonstrate that dose-response relationships existed with regard to administration of saccharin or sucrose to mice for 35 days, and this association involved testis-expressed sweet-tasting molecules (taste receptor type 1 subunit 3 [T1R3]; G protein alpha-gustducin [Galpha]). Mouse body weights and testis weights in middle- and low-dose saccharin-treated groups were increased with up-expressions of molecules involved in testicular sweet taste and steroidogenic (middle saccharin: steroidogenic acute regulatory protein [StAR]; P450 cholesterol side-chain cleavage enzyme [CYP11A1]; 17-alpha-hydroxylase/C17,20-lyase [CYP17A1]; low saccharin: StAR). Moreover, a high-dose saccharin-related decline in reproductive hormone levels and injuries to testis and sperm were observed to be associated with suppression of testicular T1R3 and Galpha, as well as steroidogenic-related factors (StAR; 3-beta-hydroxysteroid dehydrogenase [3-beta-HSD]; CYP11A1; CYP17A1; 17-beta-hydroxysteroid dehydrogenase [17-beta-HSD]), and activation of cleaved caspase-3. However, abnormalities of the testis and sperm in high- and middle-dose sucrose-exposed mice were related to the increased-cleaved caspase-3, but independent of T1R3 and/or Galpha. Collectively, our results clearly suggest that saccharin-induced physiologic effects on testis are associated with testicular T1R3 and Galpha, which differed from sucrose. We hence call for a reassessment of the excessive use of sweeteners in daily life, especially artificial ones, considering their potential side effects.

Exposure to atrazine stimulates progesterone secretion and induces oxidative stress, inflammation, and apoptosis in the ovary of pseudopregnant rats.

Atrazine (ATR) is one of the most commonly used herbicides worldwide. As an endocrine disruptor, it causes ovarian dysfunction, but the mechanism is unclear. We hypothesized that ATR could affect ovarian steroidogenesis, oxidative stress, inflammation, and apoptosis. In the current study, rats aged 28 days were treated with PMSG and HCG to obtain amounts of corpora lutea. Then, rats were injected with ATR (50 mg/kg/day) or saline (0.9%) for 7 days. Sera were collected to detect biochemical indices and progesterone (P4) level, ovaries were collected for antioxidant status, HE, qPCR, and WB analysis. Results showed that ATR exposure affected growth performance as well as serum TP, GLB, and ALB levels, increased serum P4 level and ovarian mRNA and protein levels of StAR, CYP11A1, and HSD3B. ATR treatment increased ovarian mRNA and protein levels of CREB but not PKA expression. ATR treatment increased ovarian mRNA abundances of Nrf-2 and Nqo1, MDA level, and decreased SOD, GST, and T-AOC levels. ATR exposure increased the mRNA abundances of pro-inflammatory cytokines including Tnf-α, Il-1ß, Il-6, Il-18, and Inos. ATR exposure increased the mRNA and protein level of Caspase 3 and the ratio of BAX/BCL-2. In conclusion, NRF-2/NQO1 signaling pathway and CREB might be involved in the regulation of ATR in luteal steroidogenesis, oxidative stress, inflammation, and apoptosis in rat ovary.

[Single nuclear polymorphisms in exon 3 of POMC gene and the association with growth traits in Hu sheep and East Friesian x Hu crossbred sheep].

Pro-opiomelancortin (POMC) plays important roles in the regulation of food intake and energy expenditure. The sheep exon 3 of gene POMC was amplified and sequenced by screening the DNA pools to select single nuclear polymorphisms and analyze the association with the growth traits. Two silent SNP mutations (g.273 T/C and g.456 G/A) in Hu sheep were identified. PCR-restriction fragment length polymorphism (RFLP) was used to test the g.273 T/C and the association between the g.273 T/C polymorphism and some growth traits was analyzed in Hu sheep (n = 162) and East Friesian x Hu crossbred sheep (n=130). The results showed that three genotypes, TT, TC and CC, were detected in Hu sheep with the frequencies of 0.469, 0.438 and 0.093, respectively. Two genotypes, TT and TC, were detected in East Friesian x Hu crossbred sheep with the frequencies of 0.754 and 0.246, respectively. The association analysis showed that in Hu sheep the two-month weaning weight, four-month rump height of genotype CC and the four-month body length, cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the four- and six-month weight of genotype CC were significantly higher than those of genotypes TT and TC (P < 0.01); the four-month body height and body length of genotype CC were significantly higher than those of genotypes TT (P < 0.01) and TC (P < 0.05); the four-month cannon circumference of CC genotype was significantly higher than that of TT genotype (P < 0.01). In East Friesian x Hu crossbred sheep the two-month weaning weight, four-month weight, body height, body length, chest depth and cannon circumference of genotype TC were significantly higher than those of genotype TT (P < 0.05); the six-month weight of genotype TC was significantly higher than that of genotype CC (P < 0.01). In conclusion, the exon 3 of gene POMC was associated with growth traits, and C allele was beneficial to the increase of body weight and body size traits of sheep, which potentially afford a good foundation for further study on POMC gene as aided breeding markers for growth traits in sheep.

APN Expression in Serum and Corpus Luteum: Regulation of Luteal Steroidogenesis Is Possibly Dependent on the AdipoR2/AMPK Pathway in Goats.

Adiponectin (APN) is an essential adipokine for a variety of reproductive processes. To investigate the role of APN in goat corpora lutea (CLs), CLs and sera from different luteal phases were collected for analysis. The results showed that the APN structure and content had no significant divergence in different luteal phases both in CLs and sera; however, high molecular weight APN was dominant in serum, while low molecular weight APN was more present in CLs. The luteal expression of both AdipoR1/2 and T-cadherin (T-Ca) increased on D11 and 17. APN and its receptors (AdipoR1/2 and T-Ca) were mainly expressed in goat luteal steroidogenic cells. The steroidogenesis and APN structure in pregnant CLs had a similar model as in the mid-cycle CLs. To further explore the effects and mechanisms of APN in CLs, steroidogenic cells from pregnant CLs were isolated to detect the AMPK-mediated pathway by the activation of APN (AdipoRon) and knockdown of APN receptors. The results revealed that P-AMPK in goat luteal cells increased after incubation with APN (1 µg/mL) or AdipoRon (25 µM) for 1 h, and progesterone (P4) and steroidogenic proteins levels (STAR/CYP11A1/HSD3B) decreased after 24 h. APN did not affect the steroidogenic protein expression when cells were pretreated with Compound C or SiAMPK. APN increased P-AMPK and reduced the CYP11A1 expression and P4 levels when cells were pretreated with SiAdipoR1 or SiT-Ca, while APN failed to affect P-AMPK, the CYP11A1 expression or the P4 levels when pretreated with SiAdipoR2. Therefore, the different structural forms of APN in CLs and sera may possess distinct functions; APN might regulate luteal steroidogenesis through AdipoR2 which is most likely dependent on AMPK.

Dual role of NR4A1 in porcine ovarian granulosa cell differentiation and granulosa-lutein cell regression in vitro.

This study aimed to investigate the role of NR4A1 in forskolin (FSK)-induced granulosa cell (GC) differentiation and PGF2α-induced granulosa-lutein cell (GLC) regression. For experiment 1, primary porcine GCs were pre-cultured for 6 d before induced-differentiation by FSK with or without siNR4A1, and changes in GC proliferation, lipid droplets (LDs), and P4 level were detected. For experiment 2, the GLC model was established by FSK as in experiment 1, and then PGF2α was utilized to induce GLC regression with or without siNR4A1, changes in P4 secretion, apoptosis proteins, and associated signaling pathway members were detected. Results showed that in experiment 1, FSK up-regulated NR4A1 expression during GC differentiation and decreased GC proliferation activity, which was reversed by siNR4A1. siNR4A1 inhibited the FSK-induced decreases in Cyclin B1/D1 and CDK1/2 mRNA abundances, and increases in P21/P27 mRNA abundances, and FSK-induced LD accumulation. FSK up-regulated P4 secretion and StAR, CYP11A1 and HSD3B expression, decreased CYP19A1 expression, which were reversed by siNR4A1 except for StAR expression. In experiment 2, PGF2α induced NR4A1 expression and reduced GLC viability, which were reversed by siNR4A1. Compared with PGF2α group, the levels of P4 secretion and StAR expression were higher in PGF2α+siNR4A1 group, while CYP11A1 and HSD3B expressions held at low levels. siNR4A1 inhibited PGF2α-induced expression of apoptosis proteins (caspase3, Bax, Fas, TNFa), ATF3, and phosphorylated MAPKs (ERK1/2, P38, JNK). In summary, NR4A1 is involved in regulating porcine GC differentiation and GLC regression as well as the changes in cell proliferation, apoptosis, steroidogenesis, and MAPK pathways, which provide a theoretical basis for further understanding of the mechanism of porcine luteal formation and regression.

Impact of Cadmium and Lead Exposure on Camel Testicular Function: Environmental Contamination and Reproductive Health.

The free grazing habits of camels from various sources may cause heavy metals to bioaccumulate in their tissues and organs, possibly resulting in higher amounts of these toxic substances in their bodies over time. The aim of this study was to assess the exposure impact of lead (Pb) and cadmium (Cd) on bull camels of the Lassi breed, aged 7 to 8 years, at a site near the industrial area and another two non-industrial sites, to analyze the presence of heavy metals. Samples from three sites were collected from thirty camels (n = 10/each), soil and water (n = 30), and five different plants (n = 15/each) for analysis. Testes were collected for atomic absorption spectrometry (AAS), and hematoxylin-eosin (HE) staining. Serum samples were obtained to measure testosterone levels by radioimmunoassay (RIA). Samples were obtained from plants, soil, water, blood, serum and urine for AAS. According to the results, the testes' weight, length, width, and volume significantly decreased at the industrial site compared with the other two sites as a result of exposure to Cd and Pb. Additionally, blood testosterone concentrations were considerably lower at the industrial site, indicating a detrimental impact on testicular steroidogenesis. The histological investigation of the industrial site indicated structural disturbances, including seminiferous tubule degeneration and shedding, cellular debris in seminiferous tubules, lining epithelium depletion, and vacuolation. Elevated amounts of Cd and Pb were found at the industrial site when analyzed using water, soil, plants, testes, serum, and urine. These findings demonstrate the adverse effects of Pb and Cd exposure on camel testicular function, including decreased weight and altered steroidogenesis. These findings are essential for understanding the impact of exposure to Pb and Cd on camel reproductive function and for developing successful prevention and management plans for these exposures in this species.

Sodium Arsenite Injection Induces Ovarian Oxidative Stress and Affects Steroidogenesis in Rats.

Oxidative stress is involved in the regulation of mammalian reproduction. The present study was conducted to detect the sodium arsenite-induced oxidative stress and alterations in the structure and steroidogenesis in rat ovary. Twenty female adult rats were injected i.p. with sodium arsenite (8 mg/kg BW, T) or 0.9% saline (C) for 16 days. The oxidative stress indexes and morphology of the liver, kidney, and ovary were detected using commercial kits and HE staining, respectively. The serum progesterone and estradiol were detected by RIA, and the ovarian steroidogenic gene expressions were detected by real-time PCR. Results showed that the ovarian activities of SOD and GSH-PX decreased (P < 0.05), while the ROS activity and MDA level increased (P < 0.05) in the T group. HE staining results showed that treatment with sodium arsenite damaged the ovarian morphology, resulting in reduced large and medium follicles and increased atretic follicles. Nonetheless, neither the liver nor kidney showed evident changes in the oxidative stress indexes or morphology after sodium arsenite treatment. The serum progesterone and estradiol levels decreased (P < 0.05) with the reduced expressions in the ovarian steroidogenic genes (StAR, P450scc, and 3ß-HSD) (P < 0.05). In conclusion, sodium arsenite injection can induce ovarian oxidative stress in rats which set up an appropriate model for future studies of ovarian diseases as well as the toxic mechanism of arsenic in the reproduction.

Effect of Curcumin Supplement in Summer Diet on Blood Metabolites, Antioxidant Status, Immune Response, and Testicular Gene Expression in Hu Sheep.

In summer, the high temperature affects animal growth and reproductive performance. Curcumin is a flavonoid with anti-oxidant and anti-inflammatory effects. To evaluate the effects of dietary curcumin supplement on the blood biochemical parameters and testicular gene expressions in Hu sheep in summer, a total of 144 male Hu sheep aged four months were randomly divided into three groups (Con, Cur1, and Cur2, n = 48). Sheep in Con, Cur1, and Cur2 groups were fed a basal diet supplement with 0, 450, and 900 mg (per sheep) curcumin daily, respectively. Sheep were fed for 35 days, including a pre-feed for seven days. The results showed that the supplement with 450 mg and 900 mg curcumin increased serum free fatty acid (NEFA) and glutathione peroxidase (GPX), as well as IgA and IgM. The supplement with 450 mg curcumin increased the IgG level, while the supplement with 900 mg curcumin had a lower IgG level than the supplement with 450 mg curcumin (p < 0.05). Dietary curcumin supplement increased testicular organ index, serum testosterone level, and testicular star mRNA expression (p < 0.05). Furthermore, dietary curcumin supplement linearly inhibited testicular apoptosis with increased testicular bcl-2 mRNA expression and decreased caspase-3 mRNA expression (p < 0.05). In conclusion, dietary curcumin supplement can promote lipid metabolism, antioxidant capacity, and immune response, as well as testicular development, in Hu sheep, which provides evidence of application of curcumin in sheep production.

Heat exposure affected the reproductive performance of pregnant mice: Enhancement of autophagy and alteration of subcellular structure in the corpus luteum.

To investigate whether autophagy and subcellular changes are involved in the corpus luteum after heat exposure, a total of 30 early pregnant mice were divided equally into heat stress (HS) and non-HS (NHS) groups (n = 15). Mice in the HS group were exposed to 40.5 ±â€¯0.2 ℃ for 7 consecutive days. Ovaries were collected for immunohistochemistry (IHC), western blot (WB) analysis and transmission electron microscopy (TEM). Serum was collected to determine progesterone by RIA and uteri were collected to count the implantation sites. Results showed that heat exposure increased rectal temperature, decreased body weight and number of implantation sites. WB analysis revealed that ovarian expression of LC3B and Atg7 was up-regulated, while p62 was down-regulated in the HS group. IHC results demonstrated that ovarian staining intensity of LC3B was more intense in the HS group than that of the NHS group. LC3B was mainly localized in the granulosa cells, oocytes and luteal steroidogenic cells of the HS group. TEM results revealed double-layered separated membranes indicative of autophagosomes in the luteal steroidogenic cells of the HS group. Moreover, TEM showed that the mitochondrial cristae became dearth, structure-less, swollen after HS. Additionally, the nucleus expanded and accumulation of lipid droplets increased after HS. Results also showed that heat exposure decreased serum progesterone level and ovarian P450scc expression. These results indicate that HS enhanced autophagy and altered the subcellular structure of luteal steroidogenic cells, which may contribute to interfering with the maintenance of luteal function in early pregnant mice.

The involvement of NR4A1 and NR4A2 in the regulation of the luteal function in rats.

The nuclear receptor 4A (NR4A) members play important roles in cellular proliferation, differentiation and apoptosis. The current study first evaluate the expression of ovarian NR4A1 during different luteal stages in rats. Immature rats aged 28 days were treated with sequential Pregnant mare serum gonadotropin (PMSG) (D -2) / human chorionic gonadotropin (hCG) (D 0) to induce pseudopregnancy. Serum progesterone (P4) and ovarian expression of NR4A1 were detected by RIA and WB, respectively, at follicle stage (D 0), early (D 2), middle (D 7) and late (D 14 and D 20) luteal stages. To confirm the role of NR4A1 during the luteal regression, rats were treated with prostaglandin F2α analog (PGF) for 0-8 h on D 7 to detect the expressions of NR4A1 and NR4A2. RIA result showed that serum P4 reached highest level on D 7 and then declined. WB results showed that there were two types of NR4A1 (NR4A1-L and NR4A1-S) expressed in the ovary. The ovarian NR4A1-L decreased at the late luteal stage (D 20). However, the NR4A1-S increased at the late luteal stage (D 14). After PGF treatment on D 7, the expression of NR4A1-S increased which peaked at 0.5-1 h and then declined; while NR4A1-L expression did not change within 8 h. Real-time PCR results showed that the ovarian NR4A1 mRNA increased within 0.5 h, maintained high at 1 h and then declined. The NR4A2 mRNA expression exhibited a similar pattern to that of NR4A1 mRNA, though its abundance was not as high as NR4A1. IHC results revealed that NR4A1-L was expressed mainly in the cytoplasm of luteal steroidogenic cells, faintly expressed in the follicle theca cells, oocytes and the pericytes; while NR4A2 was primarily localized in the cytoplasm of luteal steroidogenic cells. In conclusion, all these results demonstrate that NR4A2 as well as NR4A1 might be involved in the luteal development and luteolysis in rats.

Mitigation of stress from gastric mucosal injuries by mulberry extract may occur via nitric oxide synthase signaling in mice.

Acute gastric mucosal injuries are serious clinical problems worldwide and are principally found with different types of stresses in animals. A constant challenge is to find original plant products that can combat stress. In the present study, we examined the effects of big-leaf mulberry extracts on stomach injury, and the activity of nitric oxide synthases (NOS) and total antioxidant activity (TAO) in the gastric mucosae of mice during water immersion and restraint stress (WIRS). Our data showed that WIRS-exposed mice produced several injuries and showed an enhanced iNOS, reduced eNOS activity, and decreased TAO activity in the stomach, whereas pretreatment with big-leaf mulberry extracts increased TAO activitiy. The data from our immunohistochemical study indicated that both iNOS and eNOS were expressed in parietal cells and blood vessels, while nNOS was only weakly expressed in parietal cells. In conclusion, our findings suggested that big-leaf mulberry mitigated WIRS-induced stomach injuries, and NOS signaling may play important roles in the mouse stomach during the recovery process.

Expression patterns of claudin-5 and its related signals during luteal regression in pseudopregnant rats: The enhanced effect of additional PGF treatment.

To study the expression patterns of claudin-5 and its related signals during luteal regression in rats, a sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpus luteum (CL). A total of 35 rats were treated with one PGF or two PGF at an interval of 24 h from day 7 of pseudopregnancy to induce CL regression. Serum and ovaries were collected at 0, 2, 4, 8 or 24 h after one PGF injection (1 PGF), 2 or 24 h after two PGF injections (2 PGF). The serum progesterone level was detected by RIA; the ovarian expression of claudin-5, the phosphorylations of STAT3 (p-STAT3), Akt (p-Akt), ERK1/2 (p-ERK) and p38 MAPK (p-p38) were detected by western blot, real-time PCR and IHC. Results showed that serum progesterone (P4) decreased after PGF treatment. Claudin-5 mRNA decreased at 4 h and 8 h after 1 PGF and 2 h after 2 PGF, and claudin-5 protein decreased at 4 h after 1 PGF. p-STAT3 increased at 4 h after 1 PGF and 2 h after 2 PGF. p-ERK increased at 2 h after 2 PGF. The level of p-Akt decreased at 4 h after 1 PGF. PGF treatment did not alter the phosphorylation of p38 MAPK at any time points in this study. IHC results revealed that claudin-5 was expressed in the nuclei and cytoplasm of steroidogenic cells and in the vessels, while PGF induced-p-STAT3 was expressed uniformly in the cytoplasm of luteal steroidogenic cells. In conclusion, PGF treatment decreased the expression of claudin-5 and the additional PGF treatment enhanced the decrease in claudin-5 mRNA expression and the increases in ERK1/2 and STAT3 phosphorylation in the corpus luteum of pseudopregnant rats, which will contribute new information to the further study of molecular mechanism of luteal regression.

Effects of hyper- and hypothyroidism on the development and proliferation of testicular cells in prepubertal rats.

Thyroid hormones are important in the development and regulation of testes. This study was conducted to determine the effects of hyper- and hypothyroidism on testicular development in prepubertal rats aged 20-70 days. Weaning male rats (20 days old) until day 70 age were randomly divided into four groups: control, hyperthyroid (hyper-T), hypothyroid (hypo-T) and hypothyroid treated with thyroxine (T4) (hypo-T+T4). The results indicated that thyroid hormones caused a significant effect in body and testis weights, and food and water consumption. In addition there were changes in serum concentrations of tri-iodothyronine, T4, thyroid stimulating hormone (TSH) and testosterone. Histomorphology showed a significant decrease in seminiferous tubule diameter in hyper-T compared to the other groups. Leydig cell numbers showed a significant elevation in hyper-T but not in hypo-T groups. Immunostaining indicated that TSH receptor (TSHR), thyroid hormone receptors α/ß (TRαß) and proliferating cell nuclear antigen (PCNA) have the roles in testicular development. Our findings suggest that hyper- and hypo-thyroidism regulate testicular cell proliferation and spermatogenesis in prepubertal rats, indicating that expression of TSHR, TRαß and PCNA may be regulated by thyroid hormones that are involved in testicular development; and that the administration of T4 to the hypo-T+T4 group leads to an improvement in the testicular condition.

Polymorphism of FecB gene in nine sheep breeds or strains and its effects on litter size, lamb growth and development.

Nine sheep breeds or strains, including 615 individuals were screened with forced PCR RFLP method for the FecB gene to study the polymorphism and its effects on litter size, body weight and body size. Results showed that the polymorphism frequencies of FecB gene were significantly imbalanced in these breeds or strains. The Hu sheep were all homozygous carriers of FecB gene(BB). In the Chinese Merino prolific meat strain, the genotype frequencies of BB, B+ and ++ were 51%, 30% and 19%, respectively, whereas all the other flocks had only the wild-type (++) genotype. Results within the Chinese Merino prolific meat strain showed that the mean litter size of ewes with genotype BB and B+ were 2.8 (+/-0.74) and 2.3 (+/- 0.63) (P < 0.05), whereas ++ genotype ewes had a litter size of only 1.2 (+/-0.68) (P < 0.01). At day 90 after birth, the body weights of BB/B+ genotype lambs were higher than that of ++ genotype lambs (18.6 +/- 3.70 kg, 18.0 +/- 3.71 kg vs 15.6 +/- 2.22 kg, P < 0.05). In addition, the heart girth and chest width of BB/B+ genotype lambs were significantly longer than those of the ++ lambs (P < 0.05). No significant differences were observed in either body weight or body size at day 120. Litter size at first lambing from Hu at Natural Source Conservative Region was found to be significantly higher than that from the other two regions sampled (P < 0.05). In addition to the additive effect on litter size, these findings showed for the first time that the FecB gene had a positive effect on early postnatal body growth.