“Gonads Go Mad”
Heterotrimeric G Protein and the Effects of Neonatal Stress on Hypothalmic-Pituitary-Gonadal Function in Rats
Valders High School SMART Team: Alyssa Christianson, Elizabeth Evans, Jacqueline Wenzel, Kristin Schneider, Mariah Ulness, Meagan Green, Sanne De Bruijn, Vanessa Bratz
Teacher: Joseph Kinscher
Mentor: Karin Bodensteiner, Ph.D., University of Wisconsin Stevens Point, Professor of Biology
Abstract:
Neonatal stress may permanently alter hypothalamic-pituitary-gonadal function and
accelerate the onset of puberty in female rats. Heterotrimeric G proteins, found coupled to
membrane-bound receptors on the inside of cell membranes, form a central link in cell
signaling. Inactive G proteins bind guanosine diphosphate (GDP). When a signaling molecule,
such as gonadotropin releasing hormone (GnRH), binds to membrane receptors of cells in the
anterior pituitary gland, GDP is displaced by GTP (guanosine triphosphate), and the alpha
subunit separates from the beta and gamma subunits. The alpha-GTP subunit then triggers a
cell signaling cascade. In pituitary gonadotroph cells, this cascade results in the release of
follicle stimulating hormone (FSH) and luteinizing hormone (LH). These hormones will cause
female gonads (ovaries) to release estrogen and progesterone and, if hypothalamic-pituitarygonadal function is altered, may trigger early onset of puberty in female rats. The Valders
SMART Team modeled a G Protein using 3D printing technology to study structure-function
relationships in cell signaling. Hydrophobic amino acids form the switch interface between the
alpha subunit and the beta-gamma subunits stabilizing the heterotrimeric G protein. When the
alpha subunit binds GTP, Gly199 interacts with the terminal (gamma) phosphate of GTP, and
the activated alpha subunit separates from the beta-gamma subunits resulting in cell signal
propagation. Understanding how the hypothalamic-pituitary-gonadal axis is influenced by
neonatal stress in rats may help scientists to better understand puberty onset in humans.
III. Research Methods – Two Studies
V. Results
Female Rats: Female Long-Evans rats were observed five times a day for 75 minute periods for the
Female Rats: Rats with low LG mothers begin puberty earlier than rats with
first six days postpartum. The researchers measured how often the rat pups were licked and groomed
by their mothers. The rat pups were separated into two groups: pups with High Licking and Grooming
(LG) mothers- and pups with Low LG mothers. To assess the onset of puberty in female offspring, rat
pups were examined daily from day 22 to day 35 of life to determine the day of vaginal opening. To
assay for luteinizing hormone (LH) levels in adult females, blood samples were taken from the tail vein
hourly from 9 a.m. to 2 p.m. on the day of proestrus.
high LG mothers (Fig. 6). Over time rats with low LG mothers also secreted higher
levels of luteinizing hormone than rats with high LG mothers (Fig. 7). LH stimulates
ovulation.
LH (ng/mL)
Male Rats: Male Wistar rat litters were divided into two groups. For the Maternal Separation (MS)
subgroup mothers were separated from the pups three hours a day for two weeks starting on the first
postnatal day (PND). For the control group, the mothers and pups were not disturbed except for three
bedding changes. Onset of puberty in males was measured by the separation of the prepuce (foreskin)
from the penis. To measure serum concentrations of testosterone, blood samples were taken via the
sublingual (under the tongue) vein on PND 38, 40, and 42.
Time of Day
Fig. 4
1. Inactive G proteins bind guanosine diphosphate (GDP)
(Fig. 4)
2. Hydrophobic amino acids form the switch interface
between the alpha and beta-gamma subunits (Fig. 5)
3. When a signaling molecule binds to a membrane
receptor, GDP is displaced by guanosine triphosphate
(GTP) (Fig.3 #2)
4. The terminal phosphate on GTP interacts with glycine
199 (Fig. 4), and the switch interface is destabilized
Fig. 5
5. The alpha subunit separates from the beta-gamma subunits
and binds to adenylylcyclase initiating a cell signaling cascade
(Fig.3 #3-6)
http://health.arlingtonva.us/environmental-health/rats-mice/
Gly199
GDP
Based on PDB: 1GOT
Switch Interface
GTP
GnRH
Receptor
II. Cell Response to a Signal: GnRH (Fig. 3)
1. Gonadotropin releasing hormone (GnRH) binds to
the GnRH receptor.
2. GTP replaces GDP on the inactive G protein. This
activates the G protein.
3. The Alpha subunit -separates from the Beta and
Gamma subunits and activates Adenylyl cyclase.
4. Adenylyl cyclase converts ATP into cyclic AMP
(cAMP).
5. cAMP activates Protein Kinase A, triggering a
phosphorylation cascade in the cell.
6. Protein phosphorylation results in vesicles
secreting FSH and LH.
α
GDP
GTP
Fig. 3
2
γ
G protein
(inactive)
Control
-----
MS
Postnatal Day
B)
Control
MS
Treatment Group
A) Mean testosterone levels in male
rats – control versus maternal
separation (MS) group.
B) Mean day of preputial (foreskin)
separation in male rats – control
group versus MS group.
It has been shown that neonatal stress may permanently alter hypothalamicpituitary-gonadal function; affecting the onset of puberty in rats. In female rats,
puberty is accelerated as by premature vaginal opening and elevated plasma
levels of luteinizing hormone. Conversely, in male rats, puberty is delayed, as
demonstrated by decreased postnatal levels of testosterone and delayed
preputial separation. The hypothalamus, which is affected by neonatal
stress, releases Gonadotropin releasing hormone (GnRH) that binds
to the GnRH receptor on anterior pituitary cells. Activated G
proteins then complete the cellular response by triggering
protein phosphorylation. As a result, anterior pituitary cells
secrete luteinizing hormone and follicle stimulating hormone
that target the gonads, determining the onset of puberty.
Learning how neonatal stress effects hypothalamicpituitary-gonadal function in rats may give insight into
the understanding of puberty onset in humans.
4
ATP
cAMP
5
LH
GDP
Anterior Pituitary Cell
A)
VI. Conclusion
G protein
3 (active)
β
Fig. 9
The average day of preputial separation
(Fig. 9 B) during puberty in maternally
separated rats increased in comparison to
the control rats, demonstrating a
significant delay in the onset of puberty.
Adenylyl cyclase
1
Male rats: Testosterone levels (Fig. 9 A)
increased from PND 38-42 in both groups.
The rats exposed to maternal separation
(MS) demonstrated an increase in
testosterone levels between PND 38 and
40, but testosterone levels on PND 40
were not different from controls. The
control rats demonstrated the increase in
testosterone between PND 40 and 42 and
testosterone levels were significantly
higher than MS males on PND 40.
Based on PDB: 1GOT
Fig. 2
Hormone levels during proestrus –
plasma levels of LH hormone in
adult female offspring of High or
Low LG mothers.
Testosterone (ng/mL)
Researchers are studying rats (Fig. 1) to better understand hypothalamicpituitary-gonadal axis function (Fig. 2). It has been shown that neonatal stress
permanently alters hypothalamic function, affecting the onset of puberty. For
female rats puberty is accelerated, but for male rats puberty is delayed.
Hypothalmic gonadotropin releasing-hormone (GnRH)
secretion activates G proteins that are necessary for
hypothalamic-pituitary-gonadal axis functioning. This
activation triggers a cell signaling cascade that results in
the release of follicle stimulating hormone (FSH) and
luteinizing hormone (LH) from anterior pituitary cells.
Fig. 1
These hormones target the gonads. Testes begin to produce testosterone, and
ovaries begin to produce estrogen and progesterone, triggering the onset of
puberty in males and females, respectively. Understanding how the onset of
puberty is influenced by neonatal stress in rats could help researchers to
better understand puberty onset in humans.
Onset of puberty - Day of vaginal
opening in the female offspring of
High LG and Low LG mothers.
Postnatal Day
IV. Heterotrimeric G proteins form a central link in HypothalmicPituitary-Gonadal Functioning through Cell Signaling
I. Introduction
http://www.juniordentist.com/wp-content/uploads/2012/09/Pituitary-gland-anatomy.jpg
Fig. 7
Fig. 6
Protein
Kinase A
(activated)
Valders
SMART
Team
6
FSH
References:
1.) Bodensteiner, K. J., Christianson, N., Siltumens, A., Krzykowski, J. (2014). Effects of Early Maternal
Separation on Subsequent Reproductive and Behavioral Outcomes in Male Rats. The Journal of General
Psychology, vol 141, 18 February 2014
2.) Cameron, N., Del Corpo, A., Diorio, J., McAllister, K., Sharma, S., Meaney, M. J. (2008). Maternal
Programming of Sexual Behavior and Hypothalamic-Pituitary-Gonadal Function in the Female Rat. PLoS
ONE, vol 3, 21 May 2008.
3.) Lambright, D., Sondek, J., Bohm, A., Skiba, N., Hamm, H., Sigler, P. (1996). The 2.0 A Crystal Structure of
a Heterometric G Protein. Nature, vol 379, 25 January 1996.