- Luteinizing Hormone Receptor (LHR)-Mediated Signaling Pathway - Inhibits LH-induced progesterone production in rat luteal cells, with an EC₅₀ of 0.25 μM for progesterone synthesis suppression [1]
- Steroidogenic Acute Regulatory Protein (StAR) - Downregulates StAR expression in a concentration-dependent manner in rat luteal cells [1]
- Progesterone Receptor (PR) - Binds to PR in rat uterine tissues, modulating uterine growth and differentiation (no Ki value reported) [2]

1. Inhibition of LH-Stimulated Progesterone Production in Rat Luteal Cells - Cell Source: Luteal cells isolated from pregnant rats (day 10 of gestation). - Treatment Protocol: Cells were pre-incubated with Levonorgestrel (concentrations: 0.01 μM, 0.1 μM, 0.5 μM, 1 μM) for 1 hour, followed by co-incubation with luteinizing hormone (LH, 10 ng/mL) for 24 hours. - Detection Indicators & Results: - Progesterone Level: Measured via radioimmunoassay (RIA). At 0.01 μM, Levonorgestrel inhibited LH-induced progesterone production by ~20%; at 0.1 μM, inhibition rate increased to ~45%; at 1 μM, maximum inhibition (~80%) was achieved, with an EC₅₀ of 0.25 μM [1]
- cAMP Accumulation: Detected via competitive binding assay. Levonorgestrel (1 μM) reduced LH-induced cAMP levels by ~55% compared to the LH-only group [1]
- StAR Expression: Analyzed via Western blot. Levonorgestrel (0.1–1 μM) downregulated StAR protein levels by 30–60% in a concentration-dependent manner [1]
2. Modulation of Steroidogenic Enzyme Activity in Luteal Cells - Experimental Design: Rat luteal cells were treated with Levonorgestrel (1 μM) and LH (10 ng/mL) for 12 hours; activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and cholesterol side-chain cleavage enzyme (P450scc) were measured. - Results: No significant effect on 3β-HSD or P450scc activity was observed, indicating the inhibitory effect of Levonorgestrel is specific to StAR-mediated cholesterol transport [1]

1. Regulation of Uterine Growth and Hormone Levels in Ovariectomized Rats - Animal Model: Female Sprague-Dawley (SD) rats (8-week-old), ovariectomized 7 days before the experiment to eliminate endogenous steroid interference. - Treatment Protocol: Rats were divided into 4 groups (n=6 per group): - Control group: Vehicle (0.5% carboxymethyl cellulose, CMC) via oral gavage; - Low-dose group: Levonorgestrel (0.1 mg/kg/day) dissolved in vehicle, oral gavage; - Medium-dose group: Levonorgestrel (0.5 mg/kg/day), oral gavage; - High-dose group: Levonorgestrel (1 mg/kg/day), oral gavage. - Treatment Duration: 21 consecutive days. - Detection Indicators & Results: - Uterine Weight: High-dose Levonorgestrel reduced uterine wet weight by ~35% compared to the control group; low and medium doses showed no significant effect [2]
- Plasma Progesterone Level: Measured via RIA. All Levonorgestrel groups showed a dose-dependent decrease in plasma progesterone, with the high-dose group showing a ~40% reduction [2]
- Uterine Histology: High-dose Levonorgestrel induced endometrial atrophy, with reduced glandular density and thinner endometrial epithelium [2]
2. Impact on Ovarian Follicular Development in Intact Rats - Animal Model: Intact female SD rats (10-week-old, regular estrous cycle). - Treatment Protocol: Levonorgestrel (0.5 mg/kg/day, oral gavage) for 14 days. - Results: Reduced number of mature follicles (≥2 mm in diameter) by ~50% compared to the control group; no significant effect on primordial or primary follicles [2]

1. cAMP Competitive Binding Assay for LH Signaling Pathway - Reagents: Rat luteal cell lysate, [³H]-cAMP, unlabeled cAMP standard, Levonorgestrel-treated cell supernatant. - Protocol: 1. Collect supernatant from rat luteal cells treated with Levonorgestrel (1 μM) and LH (10 ng/mL) for 4 hours; 2. Mix 100 μL of supernatant with 50 μL of [³H]-cAMP (0.1 μCi/mL) and 50 μL of cAMP-binding protein (from bovine adrenal cortex) in a 96-well plate; 3. Incubate at 4°C for 18 hours; 4. Add 100 μL of cold charcoal-dextran solution (0.5% charcoal, 0.05% dextran) to separate bound and free [³H]-cAMP; 5. Centrifuge at 3000×g for 10 minutes at 4°C; 6. Transfer 150 μL of supernatant to a scintillation vial, add 3 mL of scintillation fluid, and measure radioactivity via liquid scintillation counting. - Analysis: Calculate cAMP concentration in samples using a standard curve generated with unlabeled cAMP; compare cAMP levels between Levonorgestrel-treated and control groups [1]
2. Radioimmunoassay (RIA) for Progesterone Detection - Reagents: Anti-progesterone antibody, [¹²⁵I]-labeled progesterone, progesterone standard, Levonorgestrel-treated luteal cell supernatant. - Protocol: 1. Add 50 μL of supernatant (or progesterone standard) to a test tube, followed by 50 μL of anti-progesterone antibody and 50 μL of [¹²⁵I]-progesterone; 2. Incubate at 37°C for 2 hours, then at 4°C for 16 hours; 3. Add 1 mL of cold second antibody solution (goat anti-rabbit IgG) to precipitate antibody-bound [¹²⁵I]-progesterone; 4. Centrifuge at 2500×g for 15 minutes at 4°C, discard supernatant; 5. Measure radioactivity of the precipitate using a gamma counter. - Analysis: Determine progesterone concentration in samples using a standard curve; calculate the inhibition rate of Levonorgestrel on LH-induced progesterone production [1]

1. Isolation and Culture of Rat Luteal Cells - Protocol: 1. Sacrifice pregnant rats (day 10 of gestation) by cervical dislocation; remove ovaries and place in pre-cooled Hank’s balanced salt solution (HBSS); 2. Mince ovaries into 1 mm³ fragments, add 0.2% collagenase type I, and incubate at 37°C with gentle shaking for 30 minutes; 3. Filter the cell suspension through a 70 μm cell strainer to remove tissue debris; 4. Centrifuge at 800×g for 5 minutes at 4°C, discard supernatant; resuspend cells in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin; 5. Adjust cell density to 1×10⁶ cells/mL, seed into 24-well plates (500 μL per well), and incubate at 37°C in a 5% CO₂ incubator for 24 hours to allow cell adhesion; 6. Replace medium with serum-free DMEM, add Levonorgestrel (0.01–1 μM) and LH (10 ng/mL) as needed, and continue incubation for 24 hours; 7. Collect supernatant for progesterone and cAMP detection; lyse cells for Western blot analysis of StAR protein [1]
2. Western Blot for StAR Protein Expression - Protocol: 1. Lyse treated luteal cells with RIPA buffer containing protease inhibitors; incubate on ice for 30 minutes; 2. Centrifuge at 12,000×g for 15 minutes at 4°C, collect supernatant (total protein); 3. Determine protein concentration via BCA assay; adjust all samples to the same protein concentration (20 μg/well); 4. Mix protein samples with 5× SDS loading buffer, heat at 95°C for 5 minutes to denature proteins; 5. Load samples onto a 12% SDS-PAGE gel, run electrophoresis at 80 V for 30 minutes, then 120 V for 90 minutes; 6. Transfer proteins from gel to PVDF membrane at 300 mA for 90 minutes; 7. Block membrane with 5% non-fat milk in TBST at room temperature for 1 hour; 8. Incubate membrane with primary antibody against StAR (1:1000 dilution) at 4°C overnight; 9. Wash membrane with TBST (3 times, 10 minutes each); incubate with HRP-conjugated secondary antibody (1:5000 dilution) at room temperature for 1 hour; 10. Wash membrane with TBST (3 times, 10 minutes each); visualize bands using ECL chemiluminescence reagent; quantify band intensity using ImageJ software [1]

1. Uterine Growth Regulation Experiment in Ovariectomized Rats - Protocol: 1. Animal Preparation: 8-week-old female SD rats were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection); perform bilateral ovariectomy via dorsal incision; suture incision and monitor for 7 days to ensure recovery; 2. Grouping and Treatment: Randomly divide rats into 4 groups (n=6): - Control: 0.5% CMC (1 mL/kg) via oral gavage, once daily for 21 days; - Low-dose Levonorgestrel: 0.1 mg/kg/day, dissolved in 0.5% CMC, oral gavage, once daily for 21 days; - Medium-dose Levonorgestrel: 0.5 mg/kg/day, same solvent and route as above; - High-dose Levonorgestrel: 1 mg/kg/day, same solvent and route as above; 3. Sample Collection: On day 22, sacrifice rats by cervical dislocation; collect blood via abdominal aorta, centrifuge at 3000×g for 15 minutes to separate plasma (store at -80°C for hormone detection); remove uterus, blot dry with filter paper, weigh (record wet weight), and fix 1/3 of uterine tissue in 4% paraformaldehyde for histological analysis; 4. Detection: Measure plasma progesterone via RIA; prepare paraffin sections of uterine tissue, stain with hematoxylin-eosin (HE), and observe endometrial morphology under a light microscope [2]
2. Ovarian Follicular Development Experiment in Intact Rats - Protocol: 1. Animal Selection: 10-week-old female SD rats with regular 4-day estrous cycles (confirmed via vaginal smear for 2 consecutive cycles); 2. Treatment: Rats in the experimental group received Levonorgestrel (0.5 mg/kg/day, dissolved in 0.5% CMC) via oral gavage for 14 days; control group received vehicle only; 3. Sample Collection: On day 15, sacrifice rats, remove ovaries, fix in 4% paraformaldehyde for 24 hours; 4. Histological Analysis: Prepare 5 μm serial paraffin sections of ovaries, stain with HE; count the number of primordial follicles (<50 μm), primary follicles (50–100 μm), secondary follicles (100–200 μm), and mature follicles (>200 μm) in each ovary under a light microscope (count 5 non-overlapping fields per section) [2]

1. Overall toxicity in rats—In a 21-day oral administration study (0.1–1 mg/kg/day) in ovariectomized rats, levonorgestrel did not cause significant changes in body weight, food intake, or organ weight (liver, kidney, spleen) compared to the control group [2]
2. Biochemical indicators—No significant differences were found in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), or creatinine (Cr) levels between the levonorgestrel treatment group and the control group [2]
3. Plasma protein binding rate—Neither literature [1] nor [2] reported data on the plasma protein binding rate of levonorgestrel.

[1]. J Steroid Biochem Mol Biol. 1994 Aug;50(3-4):161-6. hyperlink: https://pubmed.ncbi.nlm.nih.gov/8049144/
[2]. Exp Anim. 2011;60(4):363-71. hyperlink: https://pubmed.ncbi.nlm.nih.gov/21791876/

1. Mechanism of action - In rat luteal cells, levonorgestrel inhibits LH-stimulated progesterone production mainly by inhibiting cAMP accumulation and downregulating StAR expression, thereby blocking cholesterol transport to mitochondria (the rate-limiting step in steroid production)[1]
- In ovariectomized rats, levonorgestrel exerts an anti-estrogenic effect on the uterus by binding to PR, resulting in reduced uterine weight and endometrial atrophy; this effect is dose-dependent, with the highest activity observed at high doses (1 mg/kg/day)[2]
2. Background of the study - Reference [1] aimed to study the effects of progestins (including levonorgestrel) on LH-mediated steroid production in luteal cells, laying the foundation for understanding the role of progestins in regulating ovarian function[1]
- Reference [2] focused on assessing the effects of levonorgestrel on rat reproductive organs (uterus and ovaries), which is relevant to the development of progestin-based contraceptives or hormone replacement therapies[2]

C25H34O3

382.53566

383.259

C, 78.49; H, 8.96; O, 12.55

86679-33-6

797-63-7 (free);13635-16-0 (hexanoate);86679-33-6 (butyrate);

3086228

Typically exists as solid at room temperature

1.11g/cm3

491.7ºC at 760 mmHg

210.7ºC

1.545

5.356

0

3

6

28

740

6

C1(=O)C=C2[C@]([H])(CC1)[C@]1([H])[C@]([H])([C@@]3([H])C(CC)(CC1)([H])[C@](OC(=O)CCC)(C#C)CC3)CC2

GPKLGCALNRZIDS-AYEDEZQKSA-N

InChI=1S/C25H34O3/c1-4-7-23(27)28-25(6-3)15-13-22-21-10-8-17-16-18(26)9-11-19(17)20(21)12-14-24(22,25)5-2/h3,16,19-22H,4-5,7-15H2,1-2H3/t19-,20+,21+,22-,24-,25-/m0/s1

[(8R,9S,10R,13S,14S,17R)-13-ethyl-17-ethynyl-3-oxo-1,2,6,7,8,9,10,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-17-yl] butanoate

Levonorgestrel butyrate; 86679-33-6; Levonorgestrel butanoate; EINECS 289-270-0; L929CBB126; 13-Ethyl-17alpha-hydroxy-18,19-dinorpregn-4-en-20-yn-3-one butyrate; DTXSID801166916; (17alpha)-13-Ethyl-17-(1-oxobutoxy)-18,19-dinorpregn-4-en-20-yn-3-one;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)