Sexual Preference in Rams:Investigating the Role of ProlactinTari TanDr. Fred StormshakDept. of Animal Science(Dr. Charles Roselli, OHSU)HHMI 2006

Male-Oriented Rams

8% of domestic ram population

http://www.nature.com/news/2001/010208/images/sheep_200.jpg

Exclusively mount other rams

Observing the Behavior

• Preference Testing:

ram

ram

ewe ewe

RAM

Biological Basis for the Behavior?• Medial preoptic area-anterior hypothalamus (MPOA-AH)

Male-oriented rams more closely resemble ewes

Hypothalamus(Medial p reoptic a rea)

Anterior pituita ry

(Frontal View)

III Ventri c le

• Ovine sexually dimorphic nucleus (oSDN)

The Presence of Prolactin

K110-14 K98-6

• MPOA prolactin over-expressed in male-oriented rams (compared to female-oriented rams)

The Prolactin Gene• Over 300 biological activities

Lactation ReproductionDevelopment of mammary gland Immune ResponseAngiogenesis (development of blood vessels)

Homeostasis

http://sheepandgoat.com/images/ewenursinglamb.jpg

Transcription and the 5’ PromoterDNA mRNA ProteinTRANSCRIPTION TRANSLATION

•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

measure of “gene expression”

DNA Amplification

• Polymerase chain reaction (PCR)

known sequence

unknown

known

Need to use a modified technique…

?

Bubble PCR - Preparation1. Obtain purified genomic DNA

2. Design “bubble”, hybridize top and bottom strands

complementary5’3’

3’5’

notcomplementary

3. Digest genomic DNA with restriction enzyme (Bam HI)

unknown

known5’3’

3’5’

1 2

4. Ligate bubble to genomic DNA fragments1

2

Bubble PCRGeneral Scheme:

bubble

promoter coding region

5’

3’

3’

5’

3’ 5’

5’ 3’

(red and blue indicate complementarity)

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 AFTERdigests ligations

Differences:-greater bubble excess-longer bubble hybridization time

digest

Successful Amplification • Product ~1100 base pairs long

Preliminary Sequence

5’ 3’

• Reads from bubble construct:

bubble coding

• Next step: obtain sequence reading from coding

5’ 3’

bubble coding

Preliminary Sequence

ATGCGTCGANCNNNCGATCGACCTGACTTAAGGANAAATTGCANTCNAGGTTAAGCGGCATANCGCGCTCAGCATNACTCTCGAANGGAGACGAGGNGNTCCNGGGAACTTNNNNCACATTTCCAAAAGNNCNGCCGAAATGACCCCTTTNNATTCNNCAAGGGAANNAGATTTTCNNTGNNCTGCNTNNACCTCCANGNAAATGGATCCNCTNCNNCCCCGGGNGGNNANNGCTCNNGNATNTTCTCAAAGTNGTCGGNGAGGAGAGTCTCGAGTTNAAAGACGNGACNTCAAAGGANCCGCATNNNNTGGTCNNNGGAAAGGACTGNCCNCATGTGATNTTCCCGGTGGGCTTTAAAAATTCCNNNCCNGCNNATGTNCGGGACTAAGACNNNGTNCGNNGNCGAGACCCNAAACTNACNNTTCNANTCCTGTGNTGACTGGNAANNNGGGCTACTTNTGGAGNCCTCCNCATGGNAATNNGTNTCTCGTCNCGNATNCGGGNNTGANACTTAATTCTTNANNCCCCANNNGNAACNCNAATGNCAACTGGNCNTTCC…

Future Research

• Sequence both strands of PCR product

• Purify DNA from our male-oriented and female-oriented rams to compare promoter DNA sequences

2 Scenarios:1. DNA sequences differ2. 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