Culture of Endometrial Explants and Peri-implantation Conceptuses to Monitor Syn

Culture of Endometrial Explants and Peri-implantation Conceptuses to Monitor Synthesis and Secretion of Proteins and Prostaglandins

P. J. Hansen and J. G. Betts
Dairy Science Dept., University of Florida, Gainesville 32611-0701 and Dept. of Biological Sciences, East Texas State University, Commerce, Texas, 75429

This method was originally developed in the laboratories of R.M. Roberts and F.W. Bazer to allow metabolic labelling of secretory proteins synthesized de novo by cultured porcine endometrium (J. Reprod. Fertil. 60:41-48, 1980). It has since been used to study secretion of proteins and prostaglandins by endometrium from the cow, ewe, mare, bitch and other species. The technique is also useful for culture of peri-implantation conceptuses and placental tissues for metabolic labelling studies and to obtain conceptus secretory proteins for biological studies.

The medium used is called Pig MEM, which is a modified minimum essential medium supplemented with non-essential amino acids, vitamins, insulin and additional glucose. For metabolic labelling studies, specific amino acids are omitted or reduced in concentration to increase incorporation of radiolabel into newly-synthesized protein. Culture is performed on a rocking platform in oxygen-rich conditions to increase gaseous exchange. For conceptuses, however, satisfactory results have been obtained with an atmosphere of 5% CO₂ in air and without use of a rocking platform (J. Reprod. Immunol. 18:271-291, 1990).

Preparation of Pig MEM
This recipe is for preparing 10 l of Pig MEM using a starting MEM formulation (cat. no. M 7270 from Sigma, St. Louis, MO or cat. no. 410-2400 from GIBCO-BRL, Grand Island, NY) that is deficient in leucine, lysine, methionine and NaHCO₃. Smaller volumes can be prepared as needed.

1. In a large vessel, add 9 l of H₂0. Dissolve enough powdered MEM to prepare 10 l. Add 30 g glucose, 15 mg methionine, 52 mg leucine and 725 mg lysine-HCl so that there will be 4, 0.1, 0.1 and 1.0 times the usual concentrations of these substances found in MEM.

2. Add 22 g NaHCO₃, 100 ml of GIBCO MEM vitamin solution (cat. no. 320-1120) or equivalent, 100 ml of GIBCO non-essential amino acids solution (cat. no. 320-1140) or equivalent and 2000 IU insulin. Adjust pH to 7.1-7.3. Bring volume to 10.0 l with water and filter-sterilize (0.2 µm) aliquots of 400 ml into autoclaved 500-ml medium bottles. Stock solutions of Pig MEM can be stored for at least 6 mo at 4 C.

4. Prepare 100 ml each of 1.50 g/l methionine, 5.20 g/l leucine, and 29.20 g/l glutamine. Dissolve each supplement in water, filter-sterilize and store each supplement separately at -20 C in sterile 12 X 75 tubes. Similarly, prepare 4.0 ml aliquots of Gibco antibiotic-antimycotic solution (ABAM; cat. no. 600-5240) or equivalent.

5. Before use of Pig MEM, supplements are added to prepare the medium for specific uses. Complete Pig MEM, which is used for cultures when metabolic labelling with amino acids is not to be performed, is prepared by adding one aliquot of leucine and methionine to a 400-ml bottle of Pig MEM. A 4 ml aliquot of ABAM is also routinely added; this can be omitted if the tissue of interest is susceptible to antibiotic cytotoxicity. Porcine and ovine endometrium have been successfully cultured with up to 8 ml of ABAM/400 ml Pig MEM. For labelling with [³H]leucine or [³⁵S]methionine, omit the corresponding amino acid supplement. Medium can be stored for at least two months after addition of supplements. An aliquot of glutamine (4 ml/400 ml medium) should be added before culture if greater than three to four weeks have elapsed since

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