Regulatory Mechanisms Underlying the Expression of Prolactin Receptor in Chicken Granulosa Cells.

Prolactin (PRL) has both pro- and anti-gonadal roles in the regulation of avian ovarian functions through its interaction with the receptor (PRLR). However, neither the pattern of expression of PRLR nor its regulatory mechanisms during follicle development have been clearly defined. The objective of the present study was to investigate mechanisms of PRLR expression in chicken granulosa cells. Levels of PRLR transcript were highest in the stroma and walls of follicles < 2 mm in diameter and progressively declined with the maturation of follicles. In preovulatory follicles, PRLR was expressed at higher levels in granulosa than theca layers. FSH exerted the greatest stimulatory effect on PRLR and StAR expression in cultured granulosa cells of the 6-8 mm follicles but this effect declined as follicles matured to F1. In contrast, LH did not alter the expression of PRLR in granulosa cells of all follicular classes but increased levels of StAR in F2 and F1 granulosa cells. Both non-glycosylated- (NG-) and glycosylated- (G-) PRL upregulated basal PRLR expression in granulosa cells of the 6-8 mm, F3 or F1 follicles but had little effect in F2 follicles. Furthermore, FSH-stimulated PRLR expression was reduced by the addition of either isoform of PRL especially in F2 granulosa cells. These results indicate that PRLR is differentially distributed and regulated by FSH or PRL variants independently or in combination in the follicular hierarchy. By using activators and inhibitors, we further demonstrated that multiple signaling pathways, including PKA, PKC, PI3K, mTOR and AMPK, are not only directly involved in, but they can also converge to modulate ERK2 activity to regulate FSH-mediated PRLR and StAR expression in undifferentiated granulosa cells. These data provide new insights into the regulatory mechanisms controlling the expression of PRLR in granulosa cells.

Expression and regulation of prolactin-like protein messenger RNA in undifferentiated chicken granulosa cells.

Prolactin-like protein (PRL-L; LOC417800) is a homolog of PRL in non-mammalian vertebrates and can act as a functional ligand of PRL receptor (PRLR). Despite its widespread expression in extrapituitary tissues, mechanisms of regulation of PRL-L in the chicken ovary remain unknown. In this study, we first examined PRL-L expression in chicken ovarian developing follicles. PRL-L transcript levels were highest (P<0.05) in follicular walls of <2mm follicles and progressively declined during follicle maturation. Undifferentiated granulosa cells of 6-8mm follicles had higher (P<0.05) PRL-L mRNA levels than differentiated granulosa cells of F3, F2 or F1 follicles. In cultured undifferentiated granulosa cells, levels of PRL-L transcript were increased (P<0.05) by follicle stimulating hormone (FSH) treatment while were not altered by the addition of luteinizing hormone (LH). In addition, 10ng/ml non-glycosylated (NG-) and 1ng/ml glycosylated (G-) PRL increased (P<0.05) but at higher levels (100 or 1000ng/ml) both showed no effects on PRL-L expression. Furthermore, 100ng/ml NG-PRL enhanced (P<0.05) FSH-induced PRL-L expression, whereas the effects of G-PRL were not significant. These results suggest that PRL-L mRNA is differentially expressed in the follicular hierarchy and its high abundance in undifferentiated granulosa cells is under the regulation of FSH or PRL variants independently or in combination. Moreover, in undifferentiated granulosa cells we also provide evidence for a positive role for PKA, PKC and PI3K signaling while a negative role for ERK2 in mediating FSH stimulation of PRL-L transcription.

Characterization and Expression of Turkey Prolactin Regulatory Element Binding in the Anterior Pituitary Gland and Pancreas During Embryogenesis.

The PRL regulatory element-binding (PREB) protein is a transcription factor that was originally cloned from the rat anterior pituitary gland and characterized as a regulator of the PRL promoter. It is also strongly expressed in several extrapituitary tissues; however, its functional role is not well understood to date. In this study, we aimed to clone and characterize the turkey PREB gene and investigate its mRNA expression in the anterior pituitary gland and pancreas during embryogenesis. Based on the conserved sequence of chicken and mammalian PREB cDNAs, a turkey PREB cDNA fragment was obtained, and after sequencing of the fragment, the 5'-and 3'-ends of mRNA were amplified and determined. To identify the PREB gene structure, polymerase chain reaction (PCR) amplification was performed. The turkey PREB gene consists of 9 exons and 8 introns, and it encodes a 411-amino-acid protein. The expression of PREB mRNA in the anterior pituitary gland was measured during embryogenesis. Levels of PREB mRNA significantly increased at embryonic day 22, with maximum levels being detected on day 25 of ontogeny, which correlated with similar changes in levels of PRL mRNA. The highest level of PREB mRNA was detected on day 19 in the pancreas. However, the highest level of insulin mRNA was detected at embryonic day 25. These results indicate that PREB may be involved in the expression of PRL mRNA in the anterior pituitary gland, whereas insulin mRNA may be expressed independently of the expression of PREB mRNA in the pancreas during embryogenesis.

Effects of Vasoactive Intestinal Polypeptide and Forskolin on mRNA Expression of Prolactin and Prolactin Regulatory Element-Binding Protein in the Anterior Pituitary Gland of Chicken Embryo and Laying Hens.

Vasoactive intestinal peptide (VIP) treatment induced mRNA expression of Prolactin (PRL) in the chicken anterior pituitary gland. VIP responsive element (VRE) of the PRL promoter was identified in the various bird species. However, transcription factor, which binds to VRE, has not yet been identified. Prolactin regulatory element-binding protein (PREB) gene cloned as a candidate transcription factor binds to VRE. Increases of mRNA levels of PRL and PREB during embryogenesis were identified. However, whether VIP affects levels of PRL and PREB mRNA during embryogenesis remains unknown. The effects of VIP and forskolin on mRNA expression of PRL and PREB in the embryonic anterior pituitary gland were assessed. Furthermore, administration of VIP to laying hens was conducted to examine the relationship between VIP and PREB mRNA expression. At day 14 of the embryonic growth stage, VIP treatment did not affect mRNA levels of either PRL or PREB, whereas forskolin treatment induced the increase of these mRNA levels. At day 20, both VIP and forskolin induced an increase of PRL and PREB mRNA levels. The administration of VIP significantly increased mRNA levels of PRL and PREB in the anterior pituitary gland of White Leghorn and Nagoya. These results indicate that the effects of VIP on PRL and PREB mRNA expression levels of VIP receptor may in turn affect PRL and PREB mRNA levels in the chicken anterior pituitary gland.

Production of cloned pigs with targeted attenuation of gene expression.

The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

Spatial-genetic structuring in a red-breasted merganser (Mergus serrator) colony in the Canadian Maritimes.

The clustering of kin is widespread across the animal kingdom and two of the primary mechanisms underlying the formation of these patterns in adult kin are (1) philopatric tendencies and (2) actively maintained kin associations. Using polymorphic microsatellites, we had set out to characterize the level of genetic-spatial organization within a colony of female red-breasted mergansers (Mergus serrator) breeding on a series of small barrier islands in Kouchibouguac National Park, NB, Canada. Additionally, using nesting data from this colony, we explored possibilities for the existence of kin associations and/or cooperative interactions between these individuals; specifically in the form of the synchronization of breeding activities (i.e., incubation initiation). Our results include: (1) the detection of broad-scale genetic structuring over the entire colony, as females nesting on separate islands were to some extent genetically distinct; (2) the detection of weak, yet significant, positive spatial autocorrelation of kin at the fine scale, but only in the more densely-populated areas of this colony; and (3) the synchrony of breeding activities among proximally nesting females, apart from any factors of relatedness. While these results confirm the existence of genetic-spatial organization within this colony, the underlying mechanisms producing such a signal are inconclusive.

Changes in post-translational modifications of prolactin during development and reproductive cycles in the chicken.

Changes in proportion of glycosylated prolactin in the anterior pituitary glands of chickens were assessed using one- and two-dimensional western blotting analysis during the perihatch stage of embryos and reproductive cycles. Multiple isoforms of prolactin were detected by one-dimensional analysis and glycosylated (G) and non-glycosylated (NG) isoforms were identified by N-glycosidase and neuraminidase treatment. Increases of ratio of G to NG isoforms were observed in both embryonic stages and reproductive cycles by the one-dimensional analysis. Although a similar tendency of increase of proportion of G prolactin was obtained, different values of proportion were observed between one-dimensional and two-dimensional analysis. Since two-dimensional analysis may better resolve isoforms differing slightly in molecular size of G prolactin, the results from two-dimensional analysis may reflect the actual proportion of prolactin isoforms. Furthermore, isoforms differing in isoelectric points were detected after N-glycosidase and neuraminidase treatment. These results indicate that prolactin may also be additionally post-translationally modified such as by phosphorylation. Thus function and biological activity of prolactin were, at least in part, regulated by post-translational modification in the various physiological stages.

Cloning of PRL and VIP cDNAs of the Java sparrow (Padda oryzivora).

Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow were cloned and sequenced. The proximal region of the PRL promoter was also identified. Java sparrow PRL was found to have 88.3, 88.3, and 89.1% sequence identity at the cDNA level to PRL of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4%), turkey (88.9%) and duck (92.0%) PRL. Based on the cDNA sequence and genomic structure of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoters. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, the region upstream of the 5' untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species.

Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct.

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.

DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling.

The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.

DHCR24 gene knockout mice demonstrate lethal dermopathy with differentiation and maturation defects in the epidermis.

Desmosterolosis is an autosomal recessive disorder due to mutations in the 3beta-hydroxysterol-Delta24 reductase (DHCR24) gene that encodes an enzyme catalyzing the conversion of desmosterol to cholesterol. To date, only two patients have been reported with severe developmental defects including craniofacial abnormalities and limb malformations. We employed mice with targeted disruption of DHCR24 to understand the pathophysiology of desmosterolosis. All DHCR24-/- mice died within a few hours after birth. Their skin was wrinkleless and less pliant, leading to restricted movement and inability to suck (empty stomach). DHCR24 gene was expressed abundantly in the epidermis of control but not of DHCR24-/- mice. Accordingly, cholesterol was not detected whereas desmosterol was abundant in the epidermis of DHCR24-/- mice. Skin histology revealed thickened epidermis with few and smaller keratohyaline granules. Aberrant expression of keratins such as keratins 6 and 14 suggested hyperproliferative hyperkeratosis with undifferentiated keratinocytes throughout the epidermis. Altered expression of filaggrin, loricrin, and involcrin were also observed in the epidermis of DHCR24-/-. These findings suggested impaired skin barrier function. Indeed, increased trans-epidermal water loss and permeability of Lucifer yellow were observed in DHCR24-/- mice. DHCR24 thus plays crucial role for skin development and its proper function.

Cloning of duck PRL cDNA and genomic DNA.

Complementary DNA (cDNA) and genomic DNA, including flanking regions of the prolactin (PRL) gene of domesticated duck, were cloned and sequenced. Duck PRL was found to have 92.0, 91.7, and 91.4% sequence identity at the cDNA level to PRL of chicken, turkey, and quail, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (93.4%), turkey (91.3%), and quail (91.3%) PRL. Mature duck PRL contains the consensus sequence for N-linked glycoslylation at position 6 which is not present in either chickens or turkeys. Thus, duck PRL is likely to be post-translationally modified differently from other avian species. Based on the cDNA sequence, the genomic structure of the gene was characterized. The duck PRL gene consists of 5 exons and 4 introns. Moreover, sequence analysis of the proximal region of duck PRL promoter revealed a high degree of similarity to that of chicken and turkey PRL promoter. These results suggest that the mechanisms, which regulate expression of the PRL gene, may be widely conserved in avian species.