Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 212-215

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 212-215

Detection of Messenger RNA for Gonadotropin-Releasing  Hormone (GnRH) but not for GnRH Receptors  in Rat Pancreas*

WANG Lei, XIE Liping, HUANG Weiquan†, ZHANG Rongqing**

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710032, China

* Supported by the National Natural Sciences Foundation of China (No. 39770388)  
**To whom correspondence should be addressed

Received: 2000-12-01

Code Number: ts01068

Abstract:   

Although gonadotropin-releasing hormone (GnRH), GnRH-like mo lecule, and GnRH receptor (GnRH-R) have been reported to exist in several tissu e s other than brain or anterior pituitary, there are no reports concerning GnRH o r GnRH-R gene expression in a normal pancreatic gland. In order to define the p r oduction of GnRH as well as GnRH-R in the pancreatic gland, we examined their g e ne expression in various developmental stages of rat pancreas using the reverse transcriptase-polymerase chain reaction (RT-PCR). GnRH mRNA transcripts were f ou nd in pancreas of male and female rats at different ages, expressing at about th e same level, whereas GnRH-R mRNA transcripts could not be detected in any rat p ancreatic gland samples. These results suggest a possible biological role of GnR H in rodent pancreas.

Key  words:   gonadotrophin-releasing hormone; gonadotrophin-releasing hormone recep tor; gene expression; pancreas; reverse transcriptase-polymerase chain react ion (RT-PCR); mRNA

 Introduction   

Gonadotropin-releasing hormone (GnRH), which is synthesized in the br a in and secreted into the anterior pituitary, plays a central role in the control  of mammalian reproduction[1,2]. GnRH and GnRH mRNA have been reported t o exist  in non-hypothalamic tissues such as ovary[3,4] , breast[5] , tes tes, and placen ta[6]. The GnRH binding site and its mRNA have also been found in extrap ituitar y tissues, including gonads and placenta[7]. These findings indicate the  b iological significance of GnRH outside the hypothalamic-pituitary-gonad axis.

Huang et al. reported the distribution of GnRH-like immunoreactive ep i thelial cells in the rat digestive system, including the exocrine portion of the  pancreas, but little is known about their properties and functions there[8 ]. Gn RH analogs are of value in treating patients with certain tumors, such as pancre atic and breast cancers[5]  because they could act directly on these horm one-de pendent tumor cells through GnRH receptors on the membrane[9]. However, it is st ill not known whether non-neoplastic, normal pancreatic gland cells also have a   specific GnRH-R similar to pituitary GnRH-R and whether GnRH autocrine or para cr ine regulation exist in the normal pancreas. There have not been any reports con cerning the expression of GnRH and GnRH-R in the mammalian digestive system .

In this study, to clarify at the molecular level the production of GnR H as well as GnRH-R in pancreatic glands, we examined gene expression of both G n RH and GnRH-R in various developmental stages of the rat pancreas, using the reverse transcriptase-polymerase chain reaction (RT-PCR), and made a semi-qua nti tative analysis.

1 Materials and Methods   

1.1 Collection of rat tissues

Eight Sprague-Dawley rats were divided into four groups: adolescent male, adult male, adolescent female and adult female. All were housed at an ambien t temperature of (22±2)°C with a 12:12 h light-dark cycle, and were given  food an d water ad libitum. Fresh tissue samples collected after decapitation of the  rat s were kept in RNA later from Ambion at  4 °C . Two specimens from each rat gr oup were collected for RNA isolation.

1.2 Total RNA preparation

Total RNA from various tissues was isolated using the RNAgents Total  RNA Isolation System Kit from Promega. The integrity of the RNA was determined by fractionation on agarose gel and staining with ethidium bromide.

1.3 RT-PCR amplification

The RNA concentration was roughly determined using the absorbance at 2 60 nm. Two microgram total RNAs from each sample were reverse transcribed into cDNA in a  total volume of 20 mL using the Reverse Transcription System Kit from Promega.

Primers specific for GnRH and GnRHR (Fig.1) were designed based on the  published rat hypothalamic GnRH and pituitary GnRH receptor cDNA sequences[10] . The primers were designed to span several putative exon-intron boundaries to a llow specific amplification of cDNA and to exclude the effect of DNA contaminati on on PCR result.

PCR reactions were carried out in the presence of 1.5 mmol/L MgCl₂, 200 mmol/ L dNTP, 1.25 U Taq DNA polymerase, 1 mmol/L primers and 2 mL RT product, accor ding to Promega' s PCR Core System II protocol. After 35 cycles (denaturation at  95 °C  for 30  s,  annealing for 60 s at 50-55 °C  depending on the primers used, extension fo r 12 0 s at  72 °C , and a final extension for 5 min at  72 °C  after the last cy cle) of amplification,  5 mL  PCR reaction mix from the  50 mL  total volume were fractioned  in 1.5% agarose gel and stained with ethidium bromide. PCR for b-actin was ru n 25 cycles in parallel to rule out the possibility of RNA degradation and 5 mL P CR reaction mix were fractioned in gel together with the GnRH PCR product using the same cDNA template. Primers for b-actin were designed according to cDNA se quence from NCBI's GenBank.

To compare the mRNA levels in different pancreatic and brain tissue sa mples, semi-quantitative PCRs were performed by analyzing densitometry results w ith UVP's GDS8000 Image and Analysis System with the data presented as the ratio  of GnRH: b-actin mRNA. PCRs for GnRH (35 cycles) and b-actin (25 cycles) we re done on two specimens from each rat group, with similar results obtained.

Hypothalamic RNA from adult rat was used as concentration control. 0.1 3, 0.50, and 2.00 mL of one hypothalamic RT product were brought to a volume of  10 mL with RT diluent that contained buffer, MgCl₂ and dNTPs concentrations iden tical to those of the original RT reaction and were amplified with the GnRH prim ers.

1.4 PCR product sequencing

The RT-PCR products from the agarose gels were extracted using the Wizard^TM  PCR Preps DNA Purification System Kit from Promega, and the DNA sequ ences of these products were determined by the dideoxy chain termination method using the AmpliTaqs Cycle Sequencing Kit (Perkin-Elmer-Cetus).

2 Results   

As shown in Fig.2, GnRH mRNA transcripts were found in rat hypothalamus as well as pancreatic glands of rats of different sexes and developmental stages. In all cases, the single 356 bp band, corresponding to the size predicted by  the GnRH primers was visualized by the ethidium bromide staining, along with a 723-bp fragment of b-actin amplified in a different tube as an internal control. The direct sequencing result further verified that the GnRH cDNA in the rat pancr eas was the same as that in the rat hypothalamus[10].

The densitometric scan using the GDS8000 System showed all the ratios of GnRH pr oduct to b-actin product were approximately the same. As  indicated in Section 1, 2 mg RNA from each specimen was used in re verse transcription and 2 mL RT product then acted as PCR template, except for the hypothalamus tests, in which 2 mL RT product was used for the b-actin amp li fication but with only 0.13 mL RT product for the GnRH amplification. The resul ts showed that GnRH mRNA was expressed at about the same level in different panc reas samples, while the hypothalamic mRNA level must be at least 10 times higher

In contrast, the GnRH-R mRNA transcript (740 bp) was detected only in the pitui t ary gland which served as a positive control. Transcripts were not found in any pancreatic gland samples from the 4 rat groups (Fig.3).

3 Discussion

While the primary site for GnRH synthesis is the hypothalamus and the primary si te for GnRH action is the pituitary, GnRH is also expressed outside of the hypot halamus and has extrapituitary activity. Demonstration of widespread action of G nRH and its agonists in various reproductive and non-reproductive tissues outsi d e of the pituitary, coupled with the identification of stereospecific GnRH bindi ng sites in gonad, placenta[11] , thymus and other tissues, raises intrig uing que stions concerning the physiological significance of these findings. Preliminary studies have shown that GnRH receptor gene expression changes in the placenta ma y account for the unique time course of human chorionic gonadotropin (hCG) secre tion during pregnancy, and that  GnRH analogs are able to modulate the steroidogenesis of cultured granulosa-lu t ein cells. While details about the functional roles of GnRH in various extrapitu itary tissues are continuously being discovered, previous research has not shown  whether GnRH is actually expressed in normal pancreas, though pancreatic cancer s were known to exhibit receptors for GnRH[12]  and pancreatic tumors are  steroid-sensitive.

The current study clearly demonstrates that GnRH mRNA can be detected in pancreas of male and female rats in different developmental stages, at about the same level, which may mean that GnRH expression in normal pancreas is not af fected much by sex hormones. The direct sequencing of PCR products confirmed the  specificity of the mRNA expression in these tissues. This is the first report t hat shows the existence of GnRH gene expression in normal rat pancreas. The low abundance of GnRH-mRNA in the pancreas, perhaps less than 10% of that in the hy p othalamus, might hinder its detection by Northern blot hybridization, which we p reviously attempted using an oligo DNA probe for GnRH-mRNA.

We could not detect mRNA for GnRH-R in the rat pancreas by RT-PCR; t he refore, it is unlikely that the rat pancreas has GnRH binding sites which are id entical to pituitary GnRH-R. The finding of mRNA for GnRH but not for GnRH-R i n rat pancreas is similar to findings reported in the mouse mammary gland[5]  . So, the pancreatic exocrine gland is similar to the mammary gland not only in tectol ogy but also in that mRNA for GnRH, not GnRH-R, is detectable in both normal ti s sues, although GnRH-R-mRNA does become detectable in the tissues after a carci nomatous transformation.

We consider two possible biological roles of GnRH in pancreas. One possibility i s that  GnRH binding sites are distributed in the alimentary tract and GnRH prod uced in the pancreas, through a paracrine mechanism, stimulated exocrine activit y of the epithelial cells in the tract. Another possible role of pancreas GnRH i s that it may have an effect on elevating the gonadotropin level in sera, as has  been reported for GnRH in milk (produced by the mammary gland) which could indu ce an elevation in sera of neonates. Since these possible biological roles of pa ncreas GnRH are still speculative, further studies, including demonstration of G nRH production and secretion, are needed to clarify the physiological significan ce of GnRH in rat pancreas.

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