Structural Biochemistry/ Kiss Gene Expression

Kisspeptins are a group of proteins that come from precursors that are approximately 145 amino acids in length that get reduced to 54 and then finally about 10, 13, or 14 amino acid carboxyl terminals and are encoded by the Kiss 1 gene and are thought to be involved in the initiation of puberty by stimulating the release of the gonadotropin-releasing hormone (GnRH). As a result, there have been several studies conducted on the nuclei of Kiss neurons in the arcuate (ARC) and anteroventral periventricular (AVPV) parts of the brains of modules such as rats.

Kisspeptins behave as ligands for the G-protein-coupled receptor, GPR54 (21-23), which is believed to regulate GnRH and gonadotropin hormone secretion. Kisspeptins also trigger the release of GnRH/LH from the hypothalamus by

Kiss1 Gene Expression in the Brain of Male and Female Rats

In an experiment conducted by researchers at the University of Washington, the National University of Córdoba, and the University of Maryland, it was discovered that female rats have a greater Kiss 1 gene expression because they have a more Kiss 1 neurons than their male counterparts. This conclusion was determined by comparing Kiss 1 mRNA levels in the AVPV and ARC in the male and female rats after subjecting them to different hormonal conditions. Through this, it was shown that female rats expressed higher levels of Kiss 1 mRNA in the AVPV, however there appeared to be no difference in the Kiss 1 expression in the ARC between male and female rats. Their research suggests that sex differences in kiss 1 gene expression in the AVPV region of the brain might play a role in regulating the GnRH/LH levels of male and female rats.

The following describes the series of experiments that were undergone in order to demonstrate that the Kiss 1 gene expression is sexually differentiated in rats:

Analyzing the sex differences in Kiss 1 Neurons in the AVPV and ARC regions of the brain

A single-labelled ISH (In Situ Hybridization), which is a technique that uses a single labeled complementary RNA or DNA strand to determine where a specific piece of RNA or DNA is located in a certain slice of tissue, was used to determine the number of neurons containing mRNA and the amount of Kiss 1 mRNA in each cell. This information was then used to compare the sexual differences in Kiss 1 neurons in the AVPV and ARC by comparing the levels of mRNA and the number of mRNA containing neurons in male and female rats.

This image depicts an example of the technique Fluorescence in Situ Hybridization that is used to locate certain DNA strands using complementary pieces of DNA strands (probes) that are labeled with fluorescent dye, and then are denatured so that they could hybridize with the DNA strands of interest.

Examining the effects of sex differences in Kiss I mRNA expression

The hormone level differences between males and destrous females can be shown by the differences in Kiss 1 mRNA expression between male and female rats during their neonatal period (time period between existing in the maternal uterus and existing completely independently). A single-labeled ISH was used to compare levels of Kiss 1 mRNA in AVPV and ARC originating from adult females that were treated with thymopoietin pentapeptide on the day they were born.

RIAs (Radioimmunoassay) of FSH and LH Hormone Levels in the Blood Plasma

The serum levels of LH (Leutenizing Hormone) and FSH (follicle Stimulating Hormone) were measured using a double antibody method and an RIA kit. The rat’s LH-I-9 and FSH-I-9 were labeled with 125-I using the chloramines-T- method with LH-RP-3 and FSH-I-9 being the controls. The chloroamines-T-method is used to label small concentrations of a certain substances by producing a very specific radioactive tracer molecule—which in this case is radioactively labeled Iodine, the element that is normally used to label peptides using this technique.

Single-Labeled ISHs and Double Labeled ISHs of Brain Sections Processed for Kiss1 ISH

The brain slices were first exposed to 4% formaldehyde, acetic anhydride, sodium citrate, sodium chloride, chloroform, dehydrated in ethanols, and then air dried to prepare for hybridization. Once the hybridization was completed, the slides were put in ribonuclease (RNAse) and were allowed to develop. The slides containing sections of the brain were also examined under double-labeled ISH in a similar fashion as the single labeled ISH and were allowed to hybridize and air-dry.

Determining the Amount of Kiss 1 mRNA for the ISHs Conducted

The single-labeled ISHs a software device counted the number of cells and the number of silver grains present on each cell, which was related to the mRNA content in each cell. When the number of silver grain clusters was 1000 times greater than the number of clusters in the background, then the cells were assumed to contain Kiss 1 (were positive for Kiss 1). In the double-labeled ISHs assays, the cells were subject to fluorescence microscopy, and a software device counted the silver grains (representing TH and mRNA). The ratios for each cell of the signal to the background was determined and a cell was defined as double-labeled if it had a signal to background ratio of greater than or equal to 3 and had at least three silver grains in it. The number of double-labeled cells made up a percentage of the total Kiss1 mRNA-expressing cells.

Sources

Alexander S. Kauffman, Michelle L. Gottsch, Juan Roa, Alisa C. Byquist, Angelena Crown, Don K. Clifton, Gloria E. Hoffman, Robert A. Steiner and Manuel Tena-Sempere. Endocrinology 2007 148: 1774-1783 originally published online Jan 4, 2007; doi:10.1210/en.2006-1540