In this article we will discuss about the screening methods for anti-fertility action of drugs in females.
Screening Methods for Anti-Ovulatory Activity:
Cupric Acetate Induced Ovulation in Rabbits:
Rabbits are reflex ovulators. They ovulate within a few hours after mating or after mechanical stimulation of vagina or sometimes even the mere presence of a male or administration of certain chemicals like cupric acetate.
In this screening method, cupric acetate is used for the induction of ovulation. The rabbit ovulates within a few hours after an i.v. injection of cupric acetate (0.3 mg/kg using 1% cupric acetate in 0.9% saline). Injection of anti-ovulatory drugs, 24 h before the induction procedure prevents ovulation.
Sexually mature female albino rabbits, weighing kg, are used for the study. Animals are kept in isolation for at least 21 days to ensure that they are not pregnant and to prevent the induction of ovulation by mating. They are then treated with the test drug and 24 h later an i.v. injection of cupric acetate is given.
The rabbits are sacrificed and the ovaries examined 18-24 h later. The total number of ovulation points on both the ovaries is recorded for each animal. Then the ovaries and uterus are excised and preserved in 10% buffered formalin and subjected to histopathological evaluation.
HCG Induced Ovulation in Rats:
Immature female albino rats do not ovulate spontaneously and do not show cyclic changes of the vaginal epithelium. Priming with human chorionic gonadotropin (HGC) induces follicular maturation, followed by spontaneous ovulation 2 days later. Injection of an anti- ovulatory drug prior to the induction procedure will prevent ovulation. This principle is used for screening potential anti-ovulatory agents.
Immature female albino rats (24-26 days old) are used for the experiment. The animals are treated with various test drugs in different dose levels. After the administration of the test drug, exogenous HCG is given to induce ovulation. After 2 days, the animals are sacrificed. Their ovaries are preserved in 10% buffered formalin and subjected to histopathological evaluation. The results are compared with the control group.
Screening Methods for Estrogenic Compounds:
1. In Vivo Methods:
A primary therapeutic use of estrogen (both in vivo and in vitro) is in contraception. The rationale for these preparations is that excess exogenous estrogen inhibits FSH and LH and thus prevents ovulation.
Assay for Water Uptake:
The principle of the assay is based on the observation that the uterus responds to estrogens by increased uptake and retention of water. A peak in the uptake is observed six hours after administration.
Ovariectomised adult animals may be used for this experiment. Although it is simpler to use immature 18-day-old mice or 22-day-old rats obtained 2 days prior to the beginning of the experiment. The animals are randomly grouped. The control group is given 0.1 ml of cottonseed oil (vehicle for estradiol) subcutaneously.
The estrogen control group is given doses ranging from 0.01-0.1 µg to establish a dose-response curve. In the initial test, the test compound is given to groups at a high and low dose. In subsequent tests it is given over a range of doses to provide the dose-response curve. All doses are given in 0.1 ml of cottonseed oil.
Five hours after treatment, the animals are killed by cervical fracture and the uteri are quickly excised. The operation is begun by a longitudinal slit through the skin of the abdomen and through the body wall. The uterus is picked up with the forceps and severed from the vagina. The uterine horns are separated from the connective tissues and are then cut at their constriction point near the ovary.
The uteri are kept moist by placing them on damp (not wet) filter paper and by covering them with damp filter paper. They are then rapidly weighed in a sensitive balance. The uteri are dried in an oven at 60°C, for 24 h and are re-weighed. The percentage increase in water over control can be calculated, and compared with the values of other groups.
Procedure for Ovariectomy:
The animals are anaesthetised with ether. A single transverse incision is made in the skin of the back. This incision can be shifted readily from one side to the other, so as to lie over each ovary in turn. A small puncture is then made over the site of the ovary, which can be seen through the abdominal wall, embedded in a pad of fat.
The top of a pair of fine forceps is introduced and the fat around the ovary is grasped, care being taken not to rupture the capsule around the ovary itself. The tip of the uterine horn is then crushed with a pair of artery forceps and the ovary together with the fallopian tube removed with a single cut using a pair of fine scissors. Usually no bleeding is observed. The muscular wound is closed by absorbable sutures and the outer skin wound is closed by nylon suture.
Four-Day Uterine Weight Assay:
This assay is based on the observation that estrogens cause an increase in protein synthesis and thus bring about an increase in uterine weight. A peak is observed after about 40 h.
Immature or ovarectomised albino mice or rats can be given the test drug intramuscularly in cottonseed oil for three consecutive days. On the fourth day animals are killed by cervical fracture, the uteri rapidly excised, and the uterine contents gently squeezed out (results are unreliable if the uterine contents are not removed).
The uteri are weighed immediately in the wet state. They are then dehydrated in an oven at 100°C for 24 h and re-weighed to obtain the dry weight increase. The log dose is plotted against the wet weight to produce a sigoid curve, and the ED50 can be determined for comparison of the test compound with estradiol.
Vaginal Opening:
This assay is based on the principle that vaginal opening occurs in immature female albino mice and rats when treated with estrogenic compounds. Complete vaginal opening is considered a sign of estrogenic activity.
Immature female animals (18-day-old mice, 21-day-old rats) are used for the study. The test and standard drugs are administered to the animals intramuscularly in cottonseed oil. The vaginal opening is observed to determine estrogenic activity.
Vagina/Cornification:
This assay is based on the fact that rats and mice exhibit a cyclical ovulation with associated changes in the secretion of hormones. This leads to changes in the vaginal epithelial cells. The estrus cycle is classified into the proestrus, estrus, metestrus and diestrus stage. Drugs with estrogenic activity change the animals from whatever stage they were into the estrus stage.
Adult female albino rats having a regular estrus cycle are used for the study Animals are treated with various test and standard drugs. Change in the vagina can be observed by taking vaginal smears and examining these for cornified cells, leucocytes and epithelial cells in the normal animals and treated animals twice daily over a period of 4 days. Any drug which changes the animals into the estrus stage skipping other stages is considered to have estrogenic activity.
Various Stages of the Estrus Cycle in Rats:
The estrus cycle is a cascade of hormonal and behavioural events, which are highly synchronised and repetitive. The short and precise estrus cycle of laboratory rats has been a useful model for reproductive studies. The laboratory rat is a spontaneous ovulating, non-seasonal, polyestrus animal. It ovulates every 4-5 days throughout the year unless interrupted by pregnancy or pseudo-pregnancy.
A century ago, the English scientist Walter Heape described the progressive stages of the estrus cycle. The cycle itself is divided into four stages, centered on the period preceding estrus, the “proestrus”, which signifies the period of follicular growth in the ovary. He termed the period succeeding estrus as “metestrus” which was a recovery period following ovulation and “diestrus” a period when the ovarian secretions from the corpus luteum prepare the uterus for implantation. The estrus cycle of a rat is usually completed in four to five days.
The cycle is roughly divided into four stages:
1. Proestrus:
This is the beginning of a new cycle. The follicles of the ovary start to mature under the influence of gonadotrophic hormones, and estrogen secretion start increasing. The vaginal smear is characterised by nucleated epithelial cells; the stage lasts for about 12 h.
2. Estrus:
In this stage the uterus is enlarged and extended due to fluid accumulation; estrogen secretion is at its peak. In the estrus stage, the smear shows presence of squamous cornified cells (hexagonal or pentagonal cells). The estrus stage is usually the period of heat and is characterised as a period of sexual receptivity, when the female allows copulation. During this stage there is increased running activity. It lasts for 12 h.
3. Metestrus:
The ovary contains corpora lutea which secrete progesterone. This stage is indicated by the presence of a mixture of cornified epithelial cells and leucocytes indicating the post-ovulatory stage and desquamation of the epithelial cells. The metestrus stage lasts for about 21 h.
4. Diestrus:
The corpus lutea regress and the declining secretion of estrogen and progesterone cause regression of the uterus. The vaginal smear shows only leucocytes. This stage is the longest phase of the estrus cycle and has a duration of about 57 h.
The Experimental Procedure for Taking Vaginal Smears:
Holding the animal on the ventral side up, a drop of normal saline is inserted into the vagina with a Pasteur pipette. Care must be taken to avoid damage or injury to the vagina so as to prevent pseudo-pregnancy. The drop of normal saline should be aspirated and replaced several times. It is then transferred to a microscope slide and allowed to dry.
The smears are fixed by placing the slide in absolute alcohol for 5 s, allowing it to dry, and staining it with a 5% aqueous methylene blue solution for 10 min. The excess stain is washed off with tap water, and the slide dried and observed using a low power microscope.
Chick Oviduct Method:
The weight of the oviduct of young chicken increases depending on the dose of natural and synthetic estrogen. This principle is used for the screening of estrogenic compounds.
Seven-day-old pullet chicks are injected subcutaneously twice daily with solutions of the test compound in various doses for six days. Doses (0.02-0.5 µg) of 17β-estradiol per animal serve as standard. Six to ten chicks are used for each dosage group. On the day after the last injection, the animals are sacrificed and the weight of the body and oviduct is determined.
2. In Vitro Methods:
Potency Assay:
This assay determines the affinity of the test compound for estrogen receptor sites in the uterus.
The uptake of titrated estradiol by immature uteri must be established. The inhibition of this uptake by pre-treatment with a test compound will then indicate the estrogenic potency of the compound.
Four immature female mice (20 days old) are killed. The uteri are quickly excised and are placed in a Krebs-Ringer phosphate buffer. Pieces of diaphragm are taken from each animal to serve as control tissue for non-specific uptake of estradiol. The uteri are divided at the cervix into two horns; this helps as one horn can be used as the control and the other for testing the compound.
The tissues are placed in vials containing 5.0 ml of Krebs- Ringer phosphate buffer, incubated, and shaken at 37°C with 95% oxygen. 5% carbon dioxide is bubbled through. The radiochemical purity of the 3H-estradiol can be checked chromatographically. Buffer solution of radioactive estradiol is made up so that each 5 ml of buffer contains 0.0016 µg of radioactive estradiol (0.25 µci). A stock solution can be made and kept refrigerated for up to 6 weeks.
The excised tissues are treated as follows:
Control:
Four pieces of diaphragm are incubated and shaken with 5 ml of buffer solution for 15 min. at 37°C and are then shaken for 1 h with 5 ml of buffer containing the radioactive estradiol and 2% w/v bovine albumin.
Experimental:
Four uterine horns are incubated and are shaken in 5 ml of buffer at 37°C for 15 min. They are then incubated and shaken with 5 ml of buffer containing 2% of albumin and radioactive estradiol at 37°C for 1 h. Both control and experimental tissue are removed and washed with buffer at 37°C for 5 min., kept in damped filter paper and weighed. The tissues are then prepared for counting. Samples of 100 µl of the incubation solution are also taken for counting.
Treatment of Tissues for Counting:
The tissues are dried to determine constant weight and the dry weight recorded. Each piece of tissue is placed in a glass counting vial and incubated at 60°C in a shaking water bath with 0.5 ml of hyamine hydrochloride 10x until the tissue has completely dissolved. If the solution is discoloured, 50 µl of 20% hydrogen peroxide may be added. 50 µl of concentrated HCl and 15ml of phosphor solution are added to each vial.
The vials are allowed to equilibrate in the packed liquid scintillation counter, and counts are taken. Counting efficiency is determined by the addition of an internal standard. The results are expressed as disintegrations per minutes per unit of wet weight (dpm/mg). Test compounds can be incubated with the labelled estrogen. This helps in assaying their effectiveness in competing for the receptors in the uterus.
Estrogen Receptor-Binding Assay:
Estrogenic receptor-binding assay uses the principle of competitive binding of labelled and unlabelled estrogen on the estrogenic receptors. Estrogenic compounds displace the labelled estrogen in a concentration-dependent manner from the estrogen receptor.
Cytosol Preparation:
Uteri from 18-day-old female albino mice are removed and homogenised at 0°C in 1:50 (w/v) of Tris-sucrose buffer in a conical homogeniser. Human endometrium from menopausal women frozen within 2 h of hysterectomy and stored in liquid nitrogen can also be used. The frozen endometrium is pulverised and homogenised in 1:5 (w/v) of Tris-sucrose buffer. Homogenates are centrifuged for 1 h at 105,000 g.
Determination of Specific Binding in Mouse Uterus Cytosol as a Function of Steroid Concentration, Incubation Time and Temperature:
Triplicate aliquots of 125 µl of cytosol are incubated with 5 or 25 nM labelled steroid either for 2 or 4 h at 0°C or for 2 or 5 h at 25°C in the absence (total binding) or presence (non-specific binding) of a 100-fold excess of radio-inert steroid. Bound steroid is measured by dextran coated charcoal (DCC) adsorption.
DCC Adsorption Technique:
A 100 µl aliquot of incubated cytosol is stirred for 10 min. at 0°C in a micro titre plate with 100 µl of DCC suspension (0.625% dextran 80,000, 1.25% charcoal (Norit A)) and then centrifuged for 10 min. at 800 g. The concentration of bound steroid is determined by measuring the radioactivity in a 100 aliquot of supernatant.
For calculation of the relative binding affinity, the percentage of radio-ligand bound in the presence of a competitor compared to that bound in its absence is plotted against the concentration of unlabelled competing steroid.
Screening Methods for Anti-Estrogens:
1. In Vivo Methods:
Antagonism of Physiological Effects of Estrogens:
Anti-estrogenic compounds will inhibit some or all of the physiological effect of estrogen such as water uptake of uterus, uterotrophy and vaginal cornification. This principle is used for the screening of anti-estrogenic activity.
The assay techniques used for anti-estrogens are modifications of the estrogenic assays. The dose of estrogen used is that which is required to produce 50% of the maximum possible response. The test compound can be injected simultaneously or at varying times before or after the estrogen. The procedure for assays of water uptake, uterotrophy and vaginal cornification are followed as except that the test compounds are given with the estrogen.
2. In Vitro Methods:
Aromatase Inhibition:
This assay is based on the principle that some compounds which inhibit aromatase (estrogen synthase) can produce anti-estrogenic activity. Anti-estrogenic activity of compounds can be evaluated indirectly by evaluating aromatase-inhibiting ability.
Ovarian tissue from adult golden hamsters is used. The estrus cycle is monitored for at least 3 consecutive 4-day estrus cycles prior to the experiment. The experiments for evaluating inhibitor effects are performed with ovaries obtained from animals sacrificed on day 4 (proestrus) of the cycle. The ovaries are excised free from adhering fat tissue and quartered.
The quarters are transferred into plastic incubation flasks with 2 ml of Krebs-Ringer bicarbonate salt (KBR) solution (pH – 7.6) containing 8.4 mM glucose. The flasks are gassed with O2:CO2 (95%:5%), tightly closed and placed in a shaker/water bath (37°C) for incubation of the fragments. The incubation media are replaced with fresh KBR after preincubation for 1 h.
The ovaries are further incubated for 4 h in the presence or absence of inhibitors. 4-OH androstendione is used as standard in concentrations between 0.33 and 330 µM/1. At the end of the experiment, the incubation media are removed and centrifuged. In the supernatant estrogen, progesterone and testosterone are determined by radioimmunoassays. The data of control and test group is compared with suitable statistical analysis.
Screening Methods for Progestins:
1. In Vivo Methods:
Proliferation of Uterine Endometrium in Estrogen-Primed Rabbits – Clauberg – McPhail Test
Female rabbits weighing 800-1000 g are primed with estradiol. They are then administered with progestational compounds leading to the proliferation of endometrium and converted into the secretary phase. This principle is used for the screening of progestational compounds.
Female rabbits weighing 800-1000 g are primed with a daily injection of oestradiol 0.5 mcg/ml in aqueous solution. On day 7, the drug treatment is begun. The total dose is given in 5 equally divided fractions daily over 5 days. Twenty-four hours after the last injection, the animals are killed.
The uteri are dissected out and frozen sections of the middle portion of one horn is prepared and examined for histological interpretation. For interpretation of progestational proliferation of endometrium, the beginning of glandular development may be graded 1 and endometrium consisting only of glandular tissue may be graded 4.
Pregnancy Maintenance Test:
Progesterone is responsible for the maintenance of pregnancy. This principle is used for the screening of progestational compound.
Ovariectomy is done on day 5/10/15 of pregnancy in different groups of pregnant rats. The animals are treated with different test and standard drugs. Pregnant rats are killed 5/10/15 days later. An average of living foetuses at the end of the experiment is compared with the standard and the control group (without ovariectomy). The ED50 of progesterone is 5 mg/day in rat and less than 0.5 mg/day in mouse.
Carbonic Anhydrase Activity in Rabbit’s Endometrium:
There is a linear dose-response relationship between dose of progestogens and carbonic anhydrase activity in rabbit endometrium. This principle is used for the screening of progestational compounds.
Immature female albino rabbits are used in this study. The animals are primed with estradiol and administered test and standard drugs. After the drug treatment, the animals are sacrificed and their uterus removed. The endometrial extract of the uterus is evaluated for the carbonic anhydrase activity calorimetrically.
Prevention of Abortion in Oxytocin Treated Pregnant Rabbits:
Administration of oxytocin by IV to pregnant rabbits on the 30th day of pregnancy causes abortion. Prior administration of progestational compounds prevents the abortion. This principle is used for the detection and screening of progestational compounds.
Ten units of oxytocin IV are administered to pregnant rabbits on day 30 of pregnancy. Test and standard drugs in oil are injected twenty-four hours before. The control animal not receiving any drugs aborts within 2-30 min. after administration of oxytocin. Drugs which have progestational activity prevent abortion.
Deciduoma Reaction in Rats:
This study is based on the phenomenon of maternal/placental tumour formation due to progestational drugs in traumatised uterus of ovariectomised rats. This phenomenon is used for the screening of progestational compounds.
Ovariectomised adult female albino rats weighing between 150-200 g are used for the study. The rats are primed with four injections of 1 µg oestrone. This is followed by nine days of drug therapy. On day five, one uterine horn is exposed and 1 mg of histamine dihydrochloride injected into the lumen. Twenty-four hours after the last dose of drug, the animals are killed, the uterine horn cut off, weighed and histologically examined.
2. In Vitro Methods:
Progesterone Receptor-Binding Assay:
Progesterone receptor-binding assay uses the principle of competitive binding of labelled and unlabelled progesterone on progesterone receptors. Progestational compounds displace the labelled progesterone in a concentration-dependent manner from the progesterone receptor.
Human uteri obtained after hysterectomy is frozen in liquid nitrogen and stored at – 80°C until use. For cytosol preparation, uterine tissues are minced and homogenised with a homogeniser at 0-4°C in ice-cold PENG buffer composed of 10 mM KH2PO4, 10 mM K2HPO4 1.5 mM EDTA, 3 mM NaN3, 10% glycerol, pH 7.5. The homogenates are then centrifuged at 10, 5000 g at 4°C for 30 min. The supernatant is taken as cytosol.
The cytosol preparations are incubated with 3H-R5020 as radio-ligand at a concentration of 8 nmol/1 and increased concentrations (1 × 10–10 to 1 × 10–5 mol/1) of the competitor steroid overnight at 4°C. Then unbound steroids are adsorbed by incubating with 0.5 ml of DCC (0.5% carbon (Norit A), 0.05% dextran T400 in PENG buffer) for 10 min. at 4°C.
After centrifugation (10 min. at 1,500 g at 4°C), 0.5 ml of the supernatant is withdrawn and counted for radioactivity. To calculate the relative binding affinity, the percentage of radio-ligand bound in the presence of the competitor compared to that bound in its absence is plotted against the concentration of unlabelled competing steroid.
Screening Method for Anti-Progestational Activity:
Antagonisim of Physiological Effect of Progesterone:
The anti-progestational compound inhibits some or all the physiological effect of progesterones. This principle is used to screen the anti-progestational activity of drugs.
The procedures for assay of the Clauberg-McPhail test and deciduoma formation are as described for progestational activity except that the test compounds are given along with the progesterone.
Screening Method for Anti-Implantation Activity:
Female albino rats of established fertility in the proestrous or estrous stage are mated with mature male rats of established fertility (in the female: male ratio of 3:1). Each female is examined for the presence of spermatozoa in the early morning vaginal smear. The day on which this sign of mating is seen is taken as day 1 of pregnancy. The female is then separated and caged singly.
The test drug is administered orally to the animals once daily on specific days of pregnancy at different concentrations. On day 10th of pregnancy, the animals are laparotomised and the number of implants present in both the uterine horns as well as the number of corpora lutea (CL) on each ovary is counted.
The animals are allowed to complete the gestation period (usually 21-23 days) and the number of litters delivered, if any are counted. Pre-implantation loss and post-implantation loss are calculated using the following formula.
Pre-implantation loss = No. of CL on 10th day – No. of implants on 10th day
Post-implantation loss = No. of implants on 10th day – No. of litters delivered
% pre-implantation loss = No. of CL – No. of implants/No. of CL × 100
% post-implantation loss = No. of implant – No. of litters/No. of implants × 100
Procedure for Laparotomy:
The animal is anaesthetised with ether and the limbs tied to a rat board (waxed) with the ventral side up. The hairs on the area around the midline abdominal region are clipped with a curved scissor and the region cleaned with 70% alcohol. An incision of 2 cm length is made along the mid-line to expose the viscera. The superficially lying coils of ileum are lifted to expose the two uterine horns. The horns are examined for implantation sites.
Implants are visible as clear swellings on the uterine horns giving the uterine tube a beaded appearance. Embryos with a bright red dish aspect and a clear margin are considered to be healthy. Those of a dull blue colour with no clear margin and orientation with some exudates are considered resorbing. The number of implants and resorption sites per horn are counted. The ovaries, which lie on the upper end of the uterine horns, show corpora lutea as yellow spots over the surface. The number of corpora lutea present on each ovary is also noted.
After counting, the organs are replaced back. A small quantity of Neosporin powder is sprinkled over the organs to prevent any infection. The incision through the muscular layer is closed with a continuous suture using absorbable catguts. The skin layer is closed with continuous sutures using silk thread. An antiseptic, povidone iodine solution, is applied on the sutured area after wiping with 70% alcohol. The animal is maintained on light ether anaesthesia throughout the experiment. After laparotomy, the rats are transferred to a warm place till they recover from the anaesthesia.
Screening Methods for Abortifacient Activity:
Adult female albino rabbits are used for the study. The pregnancy date is counted from the date of observed mating. The existence of pregnancy may be confirmed by palpation after the 12th day of pregnancy. Intra-amniotic and intra-placental injections are administered to the rabbits under ether anaesthesia on the 20th day of pregnancy. The uterus is exposed through a mid-line incision; its various parts are identified by trans-illumination from a strong source of light and a particular site chosen for injection. Then the material is injected in 0. 1ml of solvent into the amniotic fluid or in 0.05ml of solvent into the placenta.
Alternatively, the drugs can be given through any route and duration from the 20th day of pregnancy. The effect of the drug is determined by looking for vaginal bleeding, changes in weight, abdominal palpation, and by post-mortem examination.
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