bioassay and pharmacology of ovarian hormones

1. Follicle stimulating hormone [FSH] in female stimulates ovarian follicle development. The follicle secretes estrogen when it becomes mature it is called grafian follicle. Lutenizing hormone[LH] in female acts on the mature grafian follicle to induce ovulation (LH peak). It also stimulates the Corpus lutum (derived from the ruptured grafian follicle after ovulation) to secrete progesterone. Secretion of Female Sex Hormones Hypothalamus GnRF [+] Anterior Pituitary Gland

2. Hormonal inter-relationship in the control of the female reproductive system

4. 2) Ovarian Phase The LH secretion peaks due to +ve feed back effect of estrogen secreted from the dominant follicle resulting in rupture of the developed follicle & expulsion of ovum. 3) Luteal Phase The remaining part of the ruptured follicle develops into corpus luteum, which secretes progesterone.

5. N.B. Progesterone is secreted during 9 months of pregnancy, firstly by corpus lupus then by placenta. If fertilization occurs: Fertilized ovum Secrete (hCG) Human chronic Gonadotrophins With LH activity Support corpus Lutum secrete Progesterone Relax uterus & thickness and vascularity of endometrium. i.e. prepare it for implantation of fertilized ovum & Protect it. If no fertilization occurs: Degeneration of corpus luteum sudden drop of progesteron loss of endometrium support necrosis of endometrium menstrual cycle This effect is stimulated by LH. The secreted progesterone promotes secretory function of the endometritm. Then

6. Plasma concentrations of ovarian hormones and gonadotrophis in women during normal menstrual cycle.

7. Ovarian Hormones Bioassay of Estrogens Estrogen 1) Vaginal smear method Principle: # Injection of estrogen the thickness of the vaginal wall, leucocytes cannot pass& only squamus cornified cells are identified in the vaginal smear in estrous stage. # The number of animals with +ve response is directly proportional to the estrogen dose. # The wall of vagina in diestrous stage is very thin& allows the passage of leucocytes into the vaginal lumen. So leucocytes appear in the vaginal smear.

8. Vaginal Smear Method

9. We use ovarictomized rats to avoid endogenous estrogen interference. 4 groups of animals in the diestrous stage are used (2T & 2S) Choose 2 SM doses of T estrogen (T1 &T2) & 2 SM doses of S (S1& S2) provided that: S2/S1=T2/T1 Each dose is injected in a group of animals & the vaginal smear is made on the third day & examined for the presence of squamus cornified cells. Plot a relation between log dose& % of animals with +ve response in each group of S&T. Determine relative potency (R.P) Procedures:

10. N.B. # It is an in vivo method # 4-point assay # Objective method (accurate) # No cross-over test can be done # this test can be used for screening & bioassay of estrogen. S2 T2 T1 S1 % +ve 50% response X Y Log dose R.P =X/Y

11. 2) Uterus weight method Ovarictomized or immature female rats to avoid endogenous estrogen interference. 4 groups of the animals are used. Choose 2 SM doses of S estrogen (S1& S2) & 2 SM doses of T estrogen (T1&T2) provided that: S2/S1=T2/T1 Each dose is injected in a group of animals in the 3rd, 4th,5th & 6th days after ovarectomy. Rats are killed on the 8th day & the uterus is isolated, dried& weighed. Principle: * Estrogen uterus weight of ovarectomized female rats. * The in weight is directly proportional to the dose of estrogen. Procedures: Plot a relation between log dose & % in uterus weight & determine R.P

12. N.B. * It is an in vivo test * 4-point assay * Objective method (accurate) * No cross-over test can be carried out because the animals are killed S2 T2 T1 S1 % in Uterus Weight X Y Log dose R.P =X/Y

13. Progesterone ## Endometrial proliferation method Bioassay of Progesterone # Progesterone causes proliferation of the endometrium of immature of ovarictomizes rabbits. # The degree of proliferation is directly proportional to the dose of progesterone. # Proliferation occurs through 4 phases Principle:

14. Endometrial proliferation method for Progesterone

15. Choose 2 SM doses of S progesterone( S1&S2) & 2 SM doses of T progesterone (T1&T2) provided that S2/S1=T2/T1 Each dose of progesterone is injected S.C in a group of animals daily for 5 days Compare the degree of proliferation in T&S We use immature or ovarictomized female rabbits to avoid interference by endogenous progesterone. Bring 4 groups of animals Inject all animals with estrogen for 6 days (for development of uterus to prepare it for progesterone action) Kill the animal, isolate the uterus & make section through it Plot a relation between log dose& degree of proliferation the determine R.P. Procedures:

16. N.B. # It is an in vivo test. # 4-point assay. # No cross-over test can be carried out because the animals are killed. S2 T2 T1 S1 Degree of Proliferation X Y Log dose R.P =X/Y

17. Prepared by: Lina Adel 911 Margret Magdi 916 Maged Melad 912 Marleen Micheal 917 Magy Magdi 914 Mary Badry 919 Magda Mahmoud 913 Marleen Melad 918 Martina Nady 915 Mary Bhgt 920 Thank You