Lihua Lv

Evidence Supporting a Role for Cocaine- and Amphetamine-Regulated Transcript (CARTPT) in Control of Granulosa Cell Estradiol Production Associated with Dominant Follicle Selection in Cattle

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

Cocaine- and Amphetamine-Regulated Transcript Accelerates Termination of Follicle-Stimulating Hormone-Induced Extracellularly Regulated Kinase 1/2 and Akt Activation by Regulating the Expression and Degradation of Specific Mitogen-Activated Protein Kinase Phosphatases in Bovine Granulosa Cells

Pleiotropic actions of cocaine- and amphetamine-regulated transcript (CART) are well described in the central nervous system and periphery, but the intracellular mechanisms mediating biological actions of CART are poorly understood. Although CART is not expressed in mouse ovaries, we have previously established CART as a novel intracellular regulator of estradiol production in bovine granulosa cells. We demonstrated that inhibitory actions of CART on estradiol production are mediated through inhibition of FSH-induced cAMP accumulation, Ca(2+) influx, and aromatase mRNA expression via a G(o/i)-dependent pathway. We also reported that FSH-induced estradiol production is dependent on Erk1/2 and Akt signaling, and CART may regulate other signaling proteins downstream of cAMP essential for estradiol production. Here, we demonstrate that CART is a potent inhibitor of FSH-stimulated Erk1/2 and Akt signaling and the mechanisms involved. Transient CART stimulation of bovine granulosa cells shortens the duration of FSH-induced Erk1/2 and Akt signaling whereas a prolonged (24 h) CART treatment blocks Erk1/2 and Akt activation in response to FSH. This CART-induced accelerated termination of Erk1/2 and Akt signaling is mediated both by induced expression and impaired ubiquitin-mediated proteasome degradation of dual specific phosphatase 5 (DUSP5) and protein phosphatase 2A. Results also support existence of a negative feedback loop in which CART via a G(o/i)-MAPK kinase dependent pathway activates Erk1/2, and the latter induces DUSP5 expression. Moreover, small interfering RNA mediated ablation of DUSP5 and/or protein phosphatase 2A prevents the CART-induced early termination of Erk1/2 and Akt signaling. Results provide novel insight into the intracellular mechanism of action of CART in regulation of FSH-induced MAPK signaling.

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation.

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.

Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/− maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/− IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

Effect of Vitamin D on basal and Luteinizing Hormone (LH) induced testosterone production and mitochondrial dehydrogenase activity in cultured Leydig cells from immature and mature rams

The objectives of this study were to investigate the potential effects of 1α,25-(OH)2VD3 (biologically active form of Vitamin D) on basal and LH-induced testosterone production and mitochondrial dehydrogenase activity in Leydig cells from immature and mature rams cultured in vitro. Leydig cells were isolated from testes of immature and mature rams, treated without (control) or with increasing concentrations of LH (1, 10, 100ng/ml) and/or 1α,25-(OH)2VD3 (1, 10, 100nM). After 24h, concentrations of testosterone in culture media were measured. After 96h, mitochondrial dehydrogenase activity in Leydig cells were measured. In immature and mature ram Leydig cells, treatment with 10 and 100ng/ml LH increased testosterone production and mitochondrial dehydrogenase activity. Treatment with 1α,25-(OH)2VD3 in the absence of LH did not increase testosterone production, but 10 and 100nM 1α,25-(OH)2VD3 increased LH induced testosterone production for both immature and mature ram Leydig cells. Treatment with all doses of 1α,25-(OH)2VD3 in the absence of LH and 10 and 100ng/ml LH in the absence of 1α,25-(OH)2VD3 increased mitochondrial dehydrogenase activity for cultured Leydig cells from immature and mature rams and 1 and 10nM 1α,25-(OH)2VD3 treatment enhanced the LH induced increase in mitochondrial dehydrogenase activity. Result demonstrate Vitamin D3 induced regulation of function of Leydig cells from immature and mature rams cultured in the presence or absence of LH and support a potential role for Vitamin D3 in regulation of gonadal function in rams.

Effect of oocyte selection, estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats.

Development of improved procedures for in vitro maturation of oocytes collected from prepubertal goats has applications for in vitro embryo production and accompanying strategies for genetic improvement. The objective of described studies was to determine the effects of oocyte grade, in vitro maturation time, antioxidant supplementation and concentrations of estradiol in the maturation medium on in vitro maturation of oocytes harvested from 1–6 mm follicles present on the ovaries (obtained from an abattoir) of 1–6-month old prepubertal Boer goats. Rates of progression to metaphase II were greater for Grade 1 oocytes (>3 compact layers of cumulus cells and evenly granulated cytoplasm) than Grade 2 oocytes (<3 layers cumulus cells and evenly granulated cytoplasm) and were lowest for Grade 3 oocytes (devoid of cumulus cells with abnormal cytoplasm). No significant effects of maturation for 24 vs 27 vs 30 h on progression of oocytes to metaphase II were observed. The addition of 5 µM β-mercaptoethanol to maturation medium increased the proportion of oocytes progressing to metaphase II and glutathione content in such oocytes, whereas supplementation with 10 mM hypotaurine was without effect. A significant inhibitory effect of in vitro maturation in the presence of high concentrations of estradiol (10 and 100 µg/mL) on progression to metaphase II was observed, and no effect was observed in response to 1 µg/mL estradiol treatment as compared with control. Results suggest that oocyte selection and β-mercaptoethanol supplementation can positively influence progression to metaphase II of oocytes harvested from ovaries of prepubertal goats, whereas high concentrations of estradiol are inhibitory to in vitro maturation.

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.

Effects of AY9944 A-7 on gonadotropin-induced meiotic resumption of oocytes and development of parthenogenetic embryos in sheep

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes.

Intrafollicular expression and potential regulatory role of cocaine- and amphetamine-regulated transcript in the ovine ovary

Follicular growth is regulated by a complex interaction of pituitary gonadotropins with local regulatory molecules. Previous studies demonstrated an important role for cocaine- and amphetamine-regulated transcript (CART) in regulation of granulosa cell estradiol production associated with dominant follicle selection in cattle. However, intraovarian expression and actions of CART in other species, including sheep, are not known. The objective of this study was to investigate the expression of CART in sheep follicles and determine the effects of CART on indices of ovine granulosa cell function linked to follicular development. Results demonstrated the expression of CART messenger RNA and prominent intraovarian localization of CART peptide in granulosa cells of sheep follicles. Granulosa cell CART messenger RNA was lower, but follicular fluid estradiol concentrations were higher in large (>5 mm) follicles vs smaller 3- to 5-mm follicles harvested from sheep ovaries of abattoir origin. CART treatment inhibited follicle stimulating hormone-induced estradiol production by cultured ovine granulosal cells and also blocked the follicle stimulating hormone-induced increase in granulosa cell numbers. Results demonstrate expression of CART in sheep follicular tissues and suggest potential biological actions of CART, which are inhibitory to ovine follicular growth and development.

Regulation of Granulosa Cell Cocaine and Amphetamine Regulated Transcript (CART) Binding and Effect of CART Signaling Inhibitor on Granulosa Cell Estradiol Production During Dominant Follicle Selection in Cattle

ABSTRACT We previously established a potential role for cocaine and amphetamine regulated transcript (CARTPT) in dominant follicle selection in cattle. CARTPT expression is elevated in subordinate versus dominant follicles, and treatment with the mature form of the CARTPT peptide (CART) decreases follicle-stimulating hormone (FSH)-stimulated granulosa cell estradiol production in vitro and follicular fluid estradiol and granulosa cell CYP19A1 mRNA in vivo. However, mechanisms that regulate granulosa cell CART responsiveness are not understood. In this study, we investigated hormonal regulation of granulosa cell CART-binding sites in vitro and temporal regulation of granulosa cell CART-binding sites in bovine follicles collected at specific stages of a follicular wave. We also determined the effect of inhibition of CART receptor signaling in vivo on estradiol production in future subordinate follicles. Granulosa cell CART binding in vitro was increased by FSH, and this induction was blocked by estrogen receptor antagonist treatment. In follicles collected in vivo at specific stages of a follicular wave, granulosa cell CART binding in the F2 (second largest), future subordinate follicle increased during dominant follicle selection. Injection into the F2 follicle (at onset of diameter deviation) of an inhibitor of the o/i subclass of G proteins (previously shown to block CART actions in vitro) resulted in increased follicular fluid estradiol concentrations in vivo. Collectively, results demonstrate hormonal regulation of granulosa cell CART binding in vitro and temporal regulation of CART binding in subordinate follicles during dominant follicle selection. Results also suggest that CART signaling may help suppress estradiol-producing capacity of the F2 (subordinate) follicle during this time period.