A possible role of a specific uterine fluid peptidase in implantation in the rat

Abstract

Although the accumulation of uterine fluid during pro-oestrus in the rat has been well documented (Long & Evans, 1922;Warren, 1938), its exact role in the reproductive process is still not clearly understood. Joshi, Yaron & Lindner (1970) isolated and characterized an endopeptidase from the uterine luminal fluid of pro-oestrous rats and from immature rats treated with oestrogen. This enzyme was later found to be unique to the uterine fluid and was not found in blood (Joshi & Murray, 1974). A protein immunologically similar to the rat endopeptidase has also been demonstrated in the uterine washings of oestradiol-treated mice of 3 different strains (Joshi & Rosenfeld, 1976). The role of this enzyme is unknown, but it has been suggested that this oestrogen-dependent peptidase may play a role in the lysis of the zona pellucida and the penetration of the ovum by the spermatozoon (Joshi & Murray, 1974).Since the loss of the zona pellucida is a prerequisite for normal implantation in the rat, the present study was undertaken in an attempt to determine whether (1) the enzyme exhibits any affinity for the zona pellucida, and (2) inhibition of the enzyme has any affect on normal implantation. MethodsWistar rats (Carworth Farms) were maintained in a temperature-controlled environment (22 + 1°C) with a 14 h light : 10 h dark cycle. Purina rat chow and tap water were freely available.The uterine peptidase was isolated and purified from rat luminal fluid by the method described by Joshi et al (1970). The protein content of the purified enzyme was measured by the procedure of Lowry, Rosebrough, Farr & Randall (1951), using bovine serum albumin (BSA) as the standard.The antiserum, prepared in rabbits from the purified peptidase as previously described (Joshi & Murray, 1974), produced one precipitin band during immunodiffusion against the immunizing antigen. The globulin fraction obtained from the antiserum and from normal rabbit serum after 40 % ammonium sulphate saturation was conjugated with fluorescein isothiocyanate (FITQ by the method of Nairn (1969). Fluorescence was observed on a Zeiss microscope using a mercury lamp with BG-12 excitation filter and appropriate secondary filters. Experiment 1. Immature female rats (22-25 days of age) were injected subcutaneously with 10 i.u. PMSG (Organon) 48-54 h before an intraperitoneal injection of 10-20 i.u. HCG (Life Sciences Pharmaceuticals). The animals were killed by cervical dislocation 12-18 h after the HCG injection. The ampullae were removed and placed in a large depression slide containing the incubation medium-a modified Krebs-Ringer-bicarbonate solution (Toyoda & Chang, 1974). The eggs in cumulus mass were dissected out of the ampullae and 1 ml of a solution of 0-1 % hyaluronidase (Sigma Chemical Co.) containing 0-1 % BSA was added. The cumulus-free eggs were placed in 1 ml incubation medium containing 0-1 ml purified uterine peptidase (1 -4 mg/ml). The eggs were incubated for 1 h at room temperature (25°C) and then washed in incubation medium, fixed in 95%ethanol, and in...

Enhancement of hatching and trophoblastic outgrowth by mouse embryos cultured in Whitten's medium containing plasmin and plasminogen

“…The identity of the zona lysin remains an enigma; it may be secreted in active form or as a proenzyme to be activated by embryonic enzymes as proposed by Denker & Hafez (1975). Joshi & Murray (1974) and Rosenfeld & Joshi (1977 have implicated an oestrogen-dependent rat uterine-specific endopeptidase in zona lysis. Hoversland & Weitlauf (1981, 1982 have observed that the incidence of zona lysis in ovariectomized mice treated with oestradiol and progesterone follows a pattern similar to changes in concentrations of a uterine chymotrypsin-like enzyme.…”

Analysis of endopeptidase and arylamidase enzymes in uterine fluid of oestrogen-treated rats

“…Proteases which are present at high levels in the uterine epithelial cells and uterine secretions during the pre-attachment stage of pregnancy include an arylamidase which hydrolyses leucine ß-naphthylamide (mouse: Bergström, 1972;rabbit: van Hoorn & Denker, 1975;Denker, 1977) and an endopeptidase which acts on the specific substrate /V-acetyl tetra-L-alanine (rat: Joshi & Murray, 1974;Rosenfeld & Joshi, 1977). The endopeptidase, cathepsin D, has also been shown to be present in high concentrations in uterine epithelial cells during early pregnancy (rat : Elangovan & Moulton, 1980) and a chymotrypsinlike amidase has been reported in uterine flushings from ovariectomized animals receiving oestrogen and progesterone (mouse: Hoversland & Weitlauf, 1978).…”

“…One approach is to administer the antisera intraluminally to the uterine horns of pregnant rats and to determine their effects on implantation (Rosenfeld & Joshi, 1977).…”

Hormone involvement in the establishment of pregnancy

“…Embryonic development beyond the unilaminar blastocyst stage does not normally proceed until the blastocyst has freed itself from the zona peUucida (McLaren, 1974). The mechanism by which the shedding of the zona peUucida is regulated is not fully understood but apparently involves the action of proteolytic enzymes arising from the endometrium (McLaren, 1970; may also be involved in the process of lysis and this activity has been implicated in the initiation of implantation (Dickmann, 1969;Owers & Blandau, 1971;Denker, 1972;Andary, Dabich & Van Winkle, 1972;Pinsker et al, 1974;Rosenfeld & Joshi, 1977). In animals in which delayed implantation occurs (e.g.…”

“…& 1972;Notides, Hamilton & Rudolph, 1973;Pinsker, Sacco & Mintz, 1974;Joshi & Murray, 1974;Rosenfeld & Joshi, 1977). These enzymes seem to be controlled by the ovarian steroids(Dickmann, 1972;Surani, 1975).…”