NBB 321 class 10

Activation of rodent female sexual behavior (continued)

Progesterone inhibition of female receptivity (ovariectomized guinea pigs)

Give EB (3.3ug) then wait 45 hrs to give P(400ug)

• It will result in 100% heat

Give EB and small P(50ug) then wait 36 hrs to give P(400ug)

• Will result in 100% heat

Give EB and medium P(400ug) then wait 20 hrs to give P(400ug)

• Will result in 50% heat

Give EB and large P(500ug) then give P(400ug) after

• Will result in 0% heat

E2, P, and positive feedback

P antagonizes (blocks) the ability of E2 to produce positive feedback when given with or preceding E2 treatment

• No heat will result if P given with E2 or right after

P can initiate an LH surge when it follows at least 12 hrs of E2 treatment

Ovariectomized Rat Model positive feedback (high LH and FSH)

If we give unopposed EB then wait 24-36 hours to give P, then we’ll get positive feedback

• Unopposed EB is EB without P in the system, only EB

Ovariectomized Rat Model Negative Feedback (high LH and FSH)

If we give small EB and P at the same time then wait to either give large E2 or P, it will continue negative feedback

General Principles of P action

If E2 has been present without P for 24 hours or more, then P facilitates female sexual receptivity

• Unopposed E2

If P is present concurrently with E2, then P inhibits female sexual receptivity

For E2 and P to induce female sexual receptivity they must be given sequentially and there must be at least 24 hrs where E2 can act without P being present

Steroid receptors during lordosis in brain cells

Lordosis posture has a circuit

• Sensory nerve located on the sides of the female senses forepaws of male

• That information gets related to the brain where it actives the spinal motor neurons

• This activation stimulates back muscle to contract and cause lordosis

Steroid receptors in the brain

E2 receptors are regionally expressed

E2 induces P receptor expression

• When E2 binds to its own receptor, it will increase P (it increases LH and FSH)

• Takes about 36 hrs for maximum P receptor expression

P binds to P receptor and causes translocation to nucleus

• Translocation takes 2-4 hrs

Presence of P blocks formation of more P receptors

There must be a period where the brain is free from P for P receptors to form

• Unopposed E2

Intracellular steroid receptors

1. Hormone passes through the cell membrane since its hydrophobic

2. It binds to its receptor, it is active and is translocated to nucleus where it binds to DNA to produce mRNA

3. mRNA is translated to create protein

4. Protein causes biological effect

Translocation of PR

Progesterone binding to its receptor can trigger a change in location of the receptor from the cytoplasm to the nucleus, where it will be transcripted and translated

Manipulating the presence of receptors

1. Can create knock-out mice

2. Temporarily eliminating receptors with antisense oligodeoxynucleotides (ODN)

Knock-out mouse

To create KO mice:

• Take embryonic stem cells with targeted gene disruptions

• Then microinject these cells in to blastocyst

• Implant injected blastocysts into uterus of pseudo-pregnant foster mother

• Mouse with tissue contribution from both blastocyst and embryonic stem cells will produce KO mice

Gene is knocked-out for all of life

For rodents, this can only be done in mice

Temporarily eliminating receptors with antisense oligodeoxynucleotides (ODN)

Anitsense ODNs are designed to have a sequence complementary to mRNA

It binds to mRNA blocking translation from occurring

• The mRNA can’t be translated into receptor protein

This decreases receptor production temporarily

Finding receptors: Autoradiography

1. Take a thin slice of tissue of area you want to study and mount it on a glass slide

2. Then incubate this slide with radioactive hormone (I132, C14, H3)

   • Radioactivity accumulates in cells with receptors (highlights where receptors are)

3. Place section facing glass slide coated with film emulsion and put it in dark for weeks to years

4. Develop film emulsion to look for cells with black dots

Autoradiography of mouse

If we take mouse and do autoradiography you could see E2 receptors since radioactive hormones accumulate there

• If you do this in knock-out mouse you will see less E2 receptors, but will still see some

• This is because there are 2 types of E2 receptors, the estrogen alpha and estrogen beta, they are only missing estrogen alpha

Estradiol and Progesterone receptor

Estradiol has its own receptors (ERa and ERB)

Estradiol induces its own receptors (activates the gene coding for ER)

• Once E2 binds to its receptor it can create more receptors

In specific brain locations, activiation of the estrogen receptor (either ERa or ERB) produces the progesterone receptor (PR)

This intracellular estrogen has two actions on receptors: inducing its own and PR

Induction of progesterone receptor by estradiol

Took autoradiography of ovariectomized mice. This is seen in mPOA of the hypothalamus

• If you give E2 over a few days to mice you can see P receptors

• If you give no E2 to KO mouse then you won’t see any P receptors, same with normal ovx mice

• If you give E2 to knockout mice then you will barely see any P receptors

E2 induction of P receptors

If you take normal rat and inject E2, then you will see P receptors

Intracellular actions of P

Some P can bind to P receptors in nucleus can change transcription

Cell surface actions of P (as a neurosteroid)

There is a second messenger system within the cell membrane

• This is a much faster process

Effect of P knockouts (PRKO) on induction of female sexual receptivity

If you don’t have P receptor, you don’t have an increase in female sexual receptivity

• No hormones result in no lordosis

• E2 results in weak lordosis

• E2 and P result in strong lordosis

Conclusion

Unopposed E2 induces P receptors, and then P induces sexual receptivity by binding to the P receptor