Sexual Preference in Rams:
Investigating the Role of
Prolactin
Tari Tan
Dr. Fred Stormshak
Dept. of Animal Science
(Dr. Charles Roselli, OHSU)
HHMI 2006
Male-Oriented Rams
Exclusively mount other
rams
8% of domestic ram
population
http://www.nature.com/news/2001/010208/images
/sheep_200.jpg
Observing the Behavior
• Preference Testing:
ram
ewe
RAM
ewe
ram
Biological Basis for the Behavior?
• Medial preoptic area-anterior hypothalamus (MPOA-AH)
(Frontal View)
III Ventricle
Hypothalamus
(Medial preoptic area)
Anterior pituitary
• Ovine sexually dimorphic nucleus (oSDN)
Male-oriented rams more closely resemble ewes
The Presence of Prolactin
• MPOA prolactin over-expressed in male-oriented rams
(compared to female-oriented rams)
K110-14
K98-6
The Prolactin Gene
• Over 300 biological activities
Lactation
Development of mammary gland
Angiogenesis (development of blood vessels)
http://sheepandgoat.com/images/ewenursinglamb.jpg
Reproduction
Immune Response
Homeostasis
Transcription and the 5’ Promoter
DNA
TRANSCRIPTION
mRNA
TRANSLATION
Protein
measure of “gene expression”
•Serves as a control for initiating transcription of mRNA
•Has been sequenced for bovine, human, and rat prolactin
genes, but not sheep
Goal: sequence the ovine 5’ promoter region
DNA Amplification
• Polymerase chain reaction (PCR)
known sequence
?
known
unknown
Need to use a modified technique…
Bubble PCR - Preparation
1. Obtain purified genomic DNA
2. Design “bubble”, hybridize top and bottom strands
complementary
5’
3’
3’
5’
not
complementary
3. Digest genomic DNA with restriction enzyme (Bam HI)
1
5’
3’
known
2
3’
5’
unknown
4. Ligate bubble to genomic DNA fragments
1
2
Bubble PCR
General Scheme:
5’
(red and blue indicate complementarity)
3’
5’
3’
3’
5’
3’
5’
promoter
coding region
bubble
Step 1: Coding primer creates complementary strand
5’
3’
3’
5’
Step 2: Once complementary strand has been synthesized, bubble primer primes
5’
3’
3’
5’
The Ligation
• Ligation of the bubble construct to digested genomic DNA
BEFORE
digests ligations
AFTER
digest
Differences:
-greater bubble excess
-longer bubble hybridization time
Successful Amplification
• Product ~1100 base pairs long
Preliminary Sequence
• Reads from bubble construct:
5’
3’
bubble
coding
• Next step: obtain sequence reading from coding
5’
3’
bubble
coding
Preliminary Sequence
ATGCGTCGANCNNNCGATCGACCTGACTTAAGGANAAATTGCANTCNAGGTTAAGCG
GCATANCGCGCTCAGCATNACTCTCGAANGGAGACGAGGNGNTCCNGGGAACTTNN
NNCACATTTCCAAAAGNNCNGCCGAAATGACCCCTTTNNATTCNNCAAGGGAANNA
GATTTTCNNTGNNCTGCNTNNACCTCCANGNAAATGGATCCNCTNCNNCCCCGGGNG
GNNANNGCTCNNGNATNTTCTCAAAGTNGTCGGNGAGGAGAGTCTCGAGTTNAAAG
ACGNGACNTCAAAGGANCCGCATNNNNTGGTCNNNGGAAAGGACTGNCCNCATGTG
ATNTTCCCGGTGGGCTTTAAAAATTCCNNNCCNGCNNATGTNCGGGACTAAGACNNN
GTNCGNNGNCGAGACCCNAAACTNACNNTTCNANTCCTGTGNTGACTGGNAANNNG
GGCTACTTNTGGAGNCCTCCNCATGGNAATNNGTNTCTCGTCNCGNATNCGGGNNTG
ANACTTAATTCTTNANNCCCCANNNGNAACNCNAATGNCAACTGGNCNTTCC…
Future Research
• Sequence both strands of PCR product
• Purify DNA from our male-oriented and femaleoriented rams to compare promoter DNA sequences
2 Scenarios:
1. DNA sequences differ
2. DNA sequences are the same
• Analyze transcription factors involved in the
regulation of the prolactin gene
Acknowledgements
HHMI
Fred Stormshak, Charles Roselli
Kevin Ahern and Indira Rajagopal
The Stormshak Lab: Caitlin, Reed, Jared, Cecily…and
the sheep