Ovine trophoblast interferon modulates the secretion of a number of proteins by ovine endometrium, but only one of these proteins has so far been identified. We examined the effects of trophoblast interferon on the secretion of matrix metalloproteinase-1, -2 and -3 by cultured ovine endometrial cells and determined whether they are mediated via effects on prostaglandin synthesis. Both ovine trophoblast interferon (30 ng ml⁻¹) and human recombinant interferon α (50 U ml⁻¹) inhibited the production of latent matrix metalloproteinase-1 and -3 (P< 0.05), as measured by enzyme assays, but had no effect on the secretion of latent matrix metalloproteinase-2. These inhibitory effects were not overcome by PGE₂ or PGF_2α (each 10 μmol l⁻¹) either alone or in combination. Indomethacin (12 μmol l⁻¹) similarly inhibited the production of latent matrix metalloproteinase-1 and -3, but production was partially restored by adding the prostaglandins either singly or in combination. PGE2 and PGF_2α together had no effect on enzyme production. These data were confirmed by gelatin and casein zymography. Northern analysis showed a 4.5-fold increase in the abundance of specific mRNA for latent matrix metalloproteinase-1 following treatment of cells with phorbol myristate acetate, but a marked decrease following interferon treatment. Thus, ovine trophoblast interferon inhibits the production of the latent forms of matrix metalloproteinase-1 and -3 by ovine endometrial cells, and this is independent of its effect on prostaglandin production.

Marmoset monkey blastocysts maintained in culture produced trophoblastic vesicles up to 4 mm in diameter that were subdivided into fragments and subcultured to produce new vesicles. These tissues are composed of an outer layer of trophoblast-like cells and an inner layer of endoderm-like cells, and resemble a blastocyst wall. When such vesicles were cultured in serum-free medium for 14 days, they increased in size but there was no significant difference in their protein content at the end of culture. The proliferation index, measured by BrdU incorporation, varied considerably within and between vesicles. The purpose of this investigation was to determine which matrix metalloproteinases are secreted by marmoset monkey trophoblastic tissue in vitro, and the effect of extracellular laminin on this secretion. It was determined by zymography that the vesicles secreted matrix metalloproteinase 2, but not matrix metalloproteinase 9, and that matrix metalloproteinase 2 was secreted as the proenzyme (72 kDa). Matrix metalloproteinases 1, 3 and 7 were not detectable in the culture medium. The addition of laminin (5–20 μg ml⁻¹), either as a substrate or in solution in the medium, did not have a consistent effect on matrix metalloproteinase 2 secretion during the culture period. The vesicles were found to express both matrix metalloproteinases 2 and 9 in both types of cell when examined by immunohistochemistry. The expression of matrix metalloproteinase 9 in the vesicles, but the absence of its secretion, indicates that specific factors, possibly of endometrial origin, may be required for inducing secretion.

Pro-protein convertases (PCs) are a family of serine proteases (furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7/8) responsible for post-translational processing and activation of inactive precursors of many regulatory proteins. Endometrial PC6 is critical for implantation in mice and for decidualization of human endometrial stromal cells (ESCs). This study investigated the endometrial expression of other PCs during the menstrual cycle and early pregnancy to elucidate potential redundancies. Furin, PC4, PACE4, and PC7 along with PC6 transcripts were detected in total endometrial RNA, whereas PC1 and PC2 transcription levels were negligible. Quantitative RT-PCR demonstrated highest levels of furin mRNA during menstruation and lowest levels during the proliferative phase. Furin protein was immunolocalized in endometrial luminal and glandular epithelia, stromal fibroblasts, endothelia, and leukocytes. PACE4 and PC7 proteins were also immunodetected in endometrial stroma and glands. Total furin, PC7, and PACE4 proteins were constitutive in both stromal and glandular compartments throughout the cycle and during first trimester pregnancy. Furthermore, Furin and PC7 transcription was unaltered during decidualization of ESCs in vitro in contrast to PC6 which is significantly up-regulated during decidualization. Thus, whereas PC6 is tightly regulated during endometrial preparation for implantation, furin, PACE4, and PC7 are constitutively expressed in human endometrium, but must be considered if PC6 is to be targeted for manipulation of fertility.

Summary. Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4–14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17β markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17β has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2α release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.

Keywords: prostaglandin synthase; sheep; endometrium; immunolocalization; mRNA; oestrogen; progesterone

Summary. A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of ⁵¹Cr and ¹²⁵I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34·2±3·0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P<0·05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46·2±1·7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically.

Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.

Ovine endometrial cells (epithelial plus stromal), prepared from ovariectomized ewes treated with oestrogen and progesterone to mimic the luteal phase of the oestrous cycle were maintained in serum-free medium for 48 h in the presence or absence of phorbol myristate acetate (PMA, 100 nmol l⁻¹), a known stimulus for production of matrix metalloproteinases (MMP) in other cells. Matrix metalloproteinase-1 (MMP-1, interstitial collagenase) and matrix metalloproteinase-2 (MMP-2, gelatinase A) activities were expressed by the cells in the absence of PMA; most were in the latent form and required activation by (4-aminophenyl) mercuric acetate (APMA). Exposure to PMA over 48 h resulted in a significant increase in MMP-1 activity but only a modest and nonsignificant increase in MMP-2 activity. Gelatin zymography demonstrated that proMMP-2 (72 kDa) was produced by both PMA-treated and untreated cells and an active form of 67 kDa was also present. Immunolocalization of MMP-1 and MMP-2 was seen within the cells following treatment with monensin. Highly purified epithelial and stromal cells were similarly cultured and analysis of the conditioned medium showed that MMP-1 and MMP-2 were produced predominantly by stromal rather than epithelial cells. Thus, both MMP-1, which degrades interstitial collagens, and MMP-2, an important enzyme for degradation of type IV and V collagens, are synthesized and released by ovine endometrial stromal cells in culture, but MMP-1 is produced primarily upon stimulation, whereas MMP-2 production is constitutive. It is postulated that these enzymes have important roles in endometrial remodelling and implantation.

Immunolocalization techniques were used to examine the distribution of the matrix metalloproteinases gelatinase B and stromelysin 1 in human endometrial specimens, taken across the normal menstrual cycle. Gelatinase B was produced by glandular epithelial cells for approximately 7 days during the proliferative phase, with polymorphonuclear leucocytes, macrophages and eosinophils providing most of this enzyme at menstruation. There was no evidence that gelatinase B is produced by stromal cells or mast cells during the cycle. Immunoreactive gelatinase B in glandular epithelial cells was greatest during the late proliferative phase and just after ovulation; its presence in glandular secretion and the uterine fluid was optimal during the peri-implantation phase. Gelatinase B was clearly associated with an influx of polymorphonuclear leucocytes, macrophages and eosinophils just before, and during, menstruation. In contrast, immunostaining for stromelysin 1 was much weaker than that for gelatinase B, and was present only around stromal cells and limited to microfocal locations at times coincident with stromal oedema (days 8–10 and 21–22). Both enzymes were widely distributed in specimens just before and during menstruation, and were particularly prominent in connective tissue stroma and vascular basement membranes. Specimens at the early proliferative stage were devoid of both enzymes. The data provide further evidence supporting a role for metalloproteinases in endometrial biology, not only in matrix remodelling during the cycle, but also in glandular secretions potentially relevant to blastocyst recognition and implantation. Our observations emphasize the functional importance of specific cell types and the temporal regulation of gelatinase B and stromelysin 1 throughout the normal menstrual cycle.

Implantation of the embryo into the endometrium is a highly regulated event that is critical for establishment of pregnancy. Molecules involved in this process provide potential targets for post-coital contraception. The aims of this study were to determine whether matrix metalloproteinases (MMPs) are present at implantation sites in rats and whether administration of a broad-based inhibitor of MMPs could inhibit embryo implantation. Uterine extracts from non-pregnant rats and from rats on days 3–9 of pregnancy were examined for the presence of MMPs. Doxycycline (5 or 15 mg day⁻¹) was administered by gavage to rats from the day of mating (day 0) to day 7 of pregnancy and the uterus was examined for implantation sites. A number of MMPs were present in all uterine samples. MMP-2 reached a peak on day 3, whereas the highest expression of MMP-7 occurred on day 7. MMP-13 and MMP-3 were present in smaller amounts. MMP-9 was detectable only on day 9. Treatment of rats with doxycycline had no effect on the number of implantation sites or on the total uterine mass. However, in treated rats, the process of decidualization was impaired and both the width and length of the decidual zone was reduced, resulting in a decrease in total decidual area from 1.20 ± 0.07 to 0.91 ± 0.07 mm² (mean ± sem, controls versus doxycycline treated, P < 0.02). It is concluded that administration of MMP inhibitors during early pregnancy retards decidual development, but does not block implantation.

Ewes actively immunized against αN, the N-terminal peptide of inhibin α₄₃ precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against αN on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against aN or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of aN. These data are consistent with a paracrine role for αN in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is essential for blastocyst implantation in mice and maintains the development of ovine embryos in culture. The expression of LIF was examined by northern blot analysis in endometrial tissue from cyclic (days 4–16) and pregnant (days 4–20) ewes, and the corresponding protein was immunolocalized. Expression of mRNA encoding LIF remained relatively constant throughout the oestrous cycle and was present during early pregnancy. A decrease in mRNA encoding LIF was observed during early pregnancy (on days 12–14) and expression was highest on days 16–20. Immunoreactive LIF was present in the cellular compartments of the endometrium throughout the oestrous cycle and early pregnancy, with maximal immunostaining in the caruncular and intercaruncular luminal epithelium, and moderate staining in the glandular epithelium and intercaruncular stroma. Immunoreactive LIF was also detected in the trophoblast cells of day 17 blastocysts. Separately cultured endometrial epithelial and stromal cells from pregnant animals both expressed mRNA encoding LIF. Ovariectomized steroid-treated ewes were studied to establish whether steroid hormones had a role in regulating endometrial LIF. Ewes treated with oestradiol alone showed lower concentrations of immunoreactive LIF in the endometrium in comparison to ovariectomized, control animals, while treatment of ovariectomized animals with both oestradiol and progesterone had a greater inhibitory effect on LIF immunolocalization. These studies demonstrate the presence of mRNA encoding LIF and protein throughout the oestrous cycle and early pregnancy and suggest that steroid hormones may be involved in their regulation.