Sham surgery was performed on mice as described above except that the ovary and tubes after being delivered from the incision site were pushed back into the peritoneum in an intact state

Sham surgery:

Tryptic hydrolysis of restrictocin and the chimeric toxins was carried out at neutral pH in 25 mM HEPES, pH 7.4 containing 1 mM CaCh, 0.5 mM EDT A. For acidic pH, the buffer containing 200 mM sodium acetate, pH 5.2 and 1 mM CaCh was used. Trypsin diluted in 50 mM PBS, pH 7.4 was added to the protein and the samples were incubated at 37 °C for specified time periods. The reaction was terminated by the addition of SDS-PAGE sample buffer. The digestion products were analyzed on a 12.5% SDS-PAGE using Tris-Giycine buffer system and restrictocin containing fragments were detected by western blotting with anti-restrictocin antibodies.

Trypsin Treatment

The culture supernates containing the assiociated hCG heterodimer as described above, were tested for their ability to inhibit hCG induced testosterone production by Leydig cells. Leydig cell suspensions were prepared from NMRI inbred adult male mice essentially by the procedure of Van Damme et al., 1974 with modifications. The procedure has been described previously Pal, 1989 and involved the measu~ement of the testosterone produced by the Leydig cells following stimulation with culture supernate containing the associated alpha and beta subunits of hCG.

Leydig cell bioassay.

Visible Spectrophotometer was from Shimadzu (Tokyo, Japan). 11Quant Microplate Reader was procured from Biotek Instruments Inc. (Winooski, VT). Gene Pulsar X Cell electroporator was purchased from Bio-Rad Laboratories (Hercules, CA) while the cuvettes for electroporating cells were obtained from BTX Harvard Apparatus Inc. (Holliston, MA). BD Calibur flow cytometer was purchased from BD Biosciences (San Jose, CA). Hu 13 Midi DNA gel electrophoresis apparatus was procured from Scie Plas (Cambridge, UK). Protean II and III polycacrylamide gel system and Mini Transblot cells were procured from Bio-Rad Laboratories (Hercules, CA). Electrophoresis Power supply EPS 500/400 was purchased from Pharmacia Biotech-AB (Uppsala, Sweden). Peltier Thermal Cycler -200 was purchased from MJ research (Waltham, MA). UVP Gel Doc-It System was purchased from UVP Bio Imaging Systems Inc. (Upland, CA). JEOL JEM-2100F field emission transmission electron microscope at the Advanced Instrumentation facility, Jawaharlal Nehru University was used to capture images. GC-MS was performed with an Agilent 7890A gas chromatography instrument coupled to an Agilent 5975C mass spectrometer and an Agilent ChemStation software (version G1701EA, Agilent Technologies, Palo Alto, CA) A HP-5MS capillary column (30m x 0.25mm i.d) coated with 0.25 11m film 5% phenyl methyl siloxane was used for separation

Forma II Series water jacketed C02 incubator for cell culture was purchased from Thermo Fischer Scientific Inc. (Waltham, MA), BOD incubator for Leishmania cultures was obtained from Jeiotech (Korea) while 37°C incubator for bacterial cultures was purchased from Thermotech Instruments (P) Ltd (Raipur, India). Gyratory incubation shaker (Ecotron) was purchased from Infors-HT (Bottmingen, Switzerland). Optiphot fluorescence microscope, Nikon E600W upright fluorescence microscope and Nikon confocal microscope C1 were from Nikon (Tokyo, Japan). FluoStar Optima and FluoStar Omega fluorescence readers were purchased from BMG Lab technologies Inc. (Offenburg, Germany). UV-160A UV

Instrumentation

Densitometry: Densitometry was performed using Labworks™ Image Acquisition and Analysis Software, UVP Biolmaging Systems, UVP Inc. (Upland, CA).

followed by several washes with PBS-Tween (50mM PBS containing 0.1 %v /v Tween 20) to remove any traces of acetic acid. Blocking: Nonspecific sites were blocked by incubating the membrane with 3%w /v milk protein (prepared in PBS-I) at 4°C overnight. Subsequently excess blocking reagent was washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-80rpm. Incubation with primary antibody: Appropriate primary antibody dilutions (usually 1:20,000; Table 3.8) were prepared in 0.1% PBS-T or 1% blocking reagent. Blots were incubated with primary antibody for 1hr at RT on a shaker set at 30-40rpm. Unbound primary antibody was then washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-80rpm. Incubation with Secondary antibody : Corresponding secondary antibody dilutions (usually 1:20,000; Table 3.8) were prepared in 0.1% PBS-I or 1% blocking reagent. Blots were incubated with secondary antibody for 1hr at RT on a shaker set at 30-40rpm. Unbound secondary antibody was then washed off by three washes of PBS-T, each for 15 min at RT on a shaker incubator set at 60-SOrpm. Detection by Enhanced Chemi-Luminescence or ECL: In the presence of hydrogen peroxide, horseradish peroxidase catalyses the oxidation of cyclic diacylhydrazides such as luminal. Following oxidation, luminal is in an excited state which decays to the ground state by emitting light. ECL reagents from EZ-ECL kit (Biological industries) were used according to manufacturer's instructions. Briefly, equal volume of ECL solution A (Luminal) and B (Hydrogen peroxide) were mixed and incubated with the membrane for approximately 5 min. Excess solution was drained off and the membrane enclosed in between two sheets of transparencies taking care to remove any air bubbles trapped in between. These were then placed in an X-ray film cassette and exposed to a sheet of X-ray film under red safety light. Depending on the antibody used, exposure times varied. Subsequently the X-ray film was transferred to developer followed by fixer solutions. Excess fixer was washed off with running water and the film air-dried.

Transfer of protein onto nitrocellulose membrane: Protein resolved by PAGE were transferred onto Hybond nitrocellulose membrane (Amersham, Pharmacia Biotech, Uppsala, Sweden) at SOV for 2.5 hours at 4°C in chilled transfer buffer (192mM glycine, 25mM Tris Base pH 8.3, 20%v lv methanol). Visualisation of protein bands transferred onto nitrocellulose membrane: Following transfer, the membrane was stained with amido black (0.1 %w lv amido black in 7%v lv acetic acid) for 30-60 s at RT. To destain, the membrane was washed in excess amounts of 7% vI v acetic acid. This was

Western Blottin