Summary. A murine pregnancy-associated protein (α1-PAP) with α1-electrophoretic mobility and an estimated molecular weight of 150 000 was present in serum from pregnant C57BL/10 mice but could not be detected in serum of mature non-pregnant females and males. During pregnancy α1-PAP was first detected on Day 7, rose to maximum levels between Days 12 and 14, and thereafter declined during the remainder of pregnancy and was undetectable by Day 8 post partum. The protein was also detected in the serum of females, but not males, subjected to an inflammatory stimulus. Examination of the α1-PAP levels during an acute-phase response in females demonstrated that the protein behaved as a typical classical acute-phase reactant, although the levels were only 10% of those observed during Days 12 to 14 of pregnancy. α1-PAP therefore appears to represent a hitherto undescribed female-specific acute phase reactant in the mouse.
Gillian T. Waites and S. C. Bell
Gillian T. Waites and S. C. Bell
Summary. Intrauterine instillation of heat-deaggregated glycogen in virgin mice induced a marked but transient luminal infiltration of polymorphonuclear leucocytes (PMNL). Similar injections of glycogen 2 days post coitum resulted in termination of pregnancy in the majority of treated females. Embryos recovered on Day 4 of pregnancy from the glycogen-treated females, before the expected time of implantation, had developed to the cavitated blastocyst stage, and were capable of undergoing characteristic outgrowth when cultured in vitro. A small proportion, however, appeared abnormal and did not develop in vitro. Cavitated blastocysts obtained from control mated females did not undergo outgrowth in vitro in the presence of intact PMNL, homogenates of these cells or a low molecular weight (<1500) fraction of the homogenates and after culture appeared similar to the abnormal blastocysts recovered from glycogen-treated uteri. It is proposed that the anti-fertility effect of PMNL infiltrates is due to a low molecular weight component of these cells which acts in utero and is cytotoxic to preimplantation blastocysts before their hatching from the zona pellucida.
S. C. Bell, Sheila Reynolds and P. J. Heald
Summary. The synthesis of uterine-soluble proteins during early pregnancy in the rat has been examined by means of dual-isotope labelling techniques and subsequent electrophoretic analysis. A protein of similar electrophoretic mobility to the uterine oestrogen-induced protein was observed, and synthesis of this 'presumptive induced protein' was maximal on Day 4 and Day 6 of pregnancy but low on Day 5. Pregnancy-associated protein synthesis was observed in many regions on polyacrylamide gels, including the β-lipoprotein, α2-macroglobulin, post-transferrin and albumin regions. Synthesis of the post-transferrin species rapidly increased from Day 4 to reach a maximum on Day 6 in the implantation tissue. The temporal pattern of synthesis of post-transferrin protein and of 'presumptive induced protein' suggests involvement in the processes of cell proliferation and decidualization.
Mohamed K Mehasseb, S C Bell and M A Habiba
We previously demonstrated that in the CD-1 mouse, which exhibits a high incidence of age-related adenomyosis, neonatal exposure to tamoxifen induced premature uterine adenomyosis and was associated with abnormal development particularly of the inner myometrium. In the present study, we examined the effect of neonatal tamoxifen administration upon uterine development in the C57/BL6J mouse strain that is not known to develop uterine adenomyosis. Female C57/BL6J pups (n=20) were treated with oral tamoxifen (1 mg/kg) from age 1 to 5 days. Uteri from control and treated mice were obtained on days 5, 10, 15 and 42 of age. We examined sections histologically using image analysis and immunohistochemistry for α-smooth muscle actin (ACTA2, α-SMA), desmin, vimentin, laminin, fibronectin and oestrogen receptor-α (ESR1). Following tamoxifen exposure, all uteri showed inner myometrium thinning, lack of continuity, disorganisation and bundling. However, adenomyosis was not seen in any uterus. ACTA2 immunostaining was less in the circular muscle layer of treated mice. The temporal pattern of desmin immunostaining found in control mice was absent in tamoxifen-treated mice. There was no difference in the localisation of laminin or fibronectin between control and tamoxifen-treated groups. However, laminin immunostaining was reduced in the circular muscle layer of treated mice. Vimentin could not be detected in either group. In conclusion, our results demonstrate that the development of the inner myometrium is particularly sensitive to oestrogen antagonism, and is affected by steroid receptor modulation. Although tamoxifen induces inner myometrial changes including that of ACTA2, desmin, ESR1 and laminin expression in C57/BL6J neonatal mice similar to those induced in CD-1 mice, C57/BL6J mice did not develop premature adenomyosis. Thus, disruption of the development and differentiation of the inner myometrium cannot alone explain the development of tamoxifen-associated adenomyosis, and this must be dependent upon its interaction with strain-dependent factors.
P. D. Rye, S. C. Bell and R. A. Walker
An epitope defined by a second generation murine monoclonal antibody (LU-BCRU-G7) produced against a breast tumour-associated fucosylated glycoprotein of M r 230 000 was found to exhibit phase specific reactivity in human endometrium during the menstrual cycle. Distribution and cycle related expression of the LU-BCRU-G7 determinant was different from the staining patterns observed with antibodies that react with the polymorphic epithelial mucins, HMFG 1 and NCRC 11, as determined by immunohistochemistry. Initial expression of the LU-BCRU-G7 determinant was associated with the peri-basal region of glandular epithelial cells with maximal staining during the mid-secretory phase. Diffuse cytoplasmic staining was also observed from day 10 to day 26, with a marked increase in the early secretory phase. Reactivity of NCRC 11 also showed maximal expression in the mid-secretory phase with no reactivity detected in early and mid-proliferative phases. The HMFG 1 defined epitope exhibited the opposite pattern of expression with maximal reactivity in the proliferative phase and little or no reactivity in the secretory phase. These findings suggest that expression of the LU-BCRU-G7, HMFG 1 and NCRC 11 defined determinants in the human glandular epithelium of the endometrium is differentially hormonally regulated, and may be of value as markers for endometrial function.
S. Sadek, T. G. Unterman and S. C. Bell
A monospecific antibody was used to determine the immunocytochemical localization of insulin-like growth factor binding protein 1 (IGFBP-1) in the rat uterus. Immunoreactive IGFBP-1 was first detected from day 5 of pregnancy in the luminal and glandular epithelium. However, immunoreactivity was most intense from day 6 in the glandular epithelium, where it was associated with apically located granules. Immunoreactive glands were located only in non-decidualized endometrium, which was limited at the implant site to a thin basal layer by growth of the antimesometrial decidua from day 7. However, glands and associated immunoreactive IGFBP-1 were prominent in the inter-implant sites until day 9, although they were detected throughout pregnancy. Similar reactivity was detected in the glands of the basal endometrium in deciduomata-bearing animals, but these decreased in number from day 7 of pseudopregnancy. No immunoreactivity was detected during the oestrous cycle but could be induced in ovariectomized animals by sequential oestradiol and oestradiol plus progesterone treatment. The observations were consistent with IGFBP-1 representing a secretory product of the glandular epithelium and could either play a role in development of the trophoblastic component of the conceptus during the pre-placental period of anti-mesometrial implantation or in the endometrium acting as an inhibitor of local IGF-I action and in either case by transporting IGF-I from the stromal to the glandular luminal environment.
Mohamed K Mehasseb, S C Bell and M A Habiba
We used a neonatal mouse model to examine the histogenesis of uterine adenomyosis, and to test whether adenomyosis is due to an abnormality in myometrial differentiation, or in extracellular matrix proteins expression. We also studied the effects of tamoxifen and estradiol on uterine development, myometrial differentiation, and organization. Female CD1 pups were treated with oral tamoxifen (1 mg/kg) (n=27) or estradiol (0.1 mg/kg) (n=24) from age 1 to 5 days. Uteri from control (n=27) and treated mice were obtained on days 2, 5, 10, 15, and 42 of age. We examined the sections histologically, using image analysis and immunohistochemistry for α-smooth muscle actin (α-SMA), desmin, vimentin, laminin, fibronectin, and estrogen receptor-α. Following tamoxifen exposure, all uteri showed adenomyosis by 6 weeks of age (seen as early as day 10). The inner myometrium showed thinning, lack of continuity, disorganization, and bundling. α-SMA expression was normal. Desmin expression normally showed a wave of maturation that was absent in tamoxifen-treated mice. In the estradiol group, adenomyosis was not observed. All uterine layers were normally developed, but hypertrophied. The inner myometrium retained its circular arrangement. There was no difference in the localization of laminin or fibronectin between groups (laminin expression was reduced in the tamoxifen treated uteri). Vimentin could not be detected in all groups. Our results suggest that the development of the inner myometrium is particularly sensitive to estrogen antagonism, and can be affected by steroid receptors modulation. Disruption of the inner myometrium may play a role in the development of uterine adenomyosis.
M.-T. Badet, S. C. Bell and W. D. Billington
Summary. Supernatants from short-term in-vitro cultures of decidual tissue, obtained from the uteri of pregnant mice from Days 4 to 13 post coitum (Day 1 = day of mating), were assessed for immunoregulatory activity by their addition to a mixed lymphocyte reaction (MLR), an in-vitro analogue of the afferent arm of the immune response. All culture supernatants tested possessed inhibitory activity in the MLR, although the extent of inhibition was affected by seeding density, length of culture, and the day of pregnancy from which decidual tissue was obtained. Inhibitory activity produced by decidual cultures increased from Day 4 to reach a maximum on Day 8, and then declined to Day 11. Two morphologically distinct cell types were present in all decidual cultures; flat dendritic cells, considered to represent decidual cells, and small round cells, but whether immunoregulatory factors are associated with both is uncertain. The results suggest that decidual tissue could fulfil a role in the local partial blockade of the afferent arm of the maternal immune response during pregnancy.
G. Correia da Silva, N. Teixeira, J. H. Pringle and S. C. Bell
The patterns of expression of mRNA encoding decidualization-associated protein (DAP) in the rat uterus from early, mid-, and late stages of pregnancy were examined by in situ hybridization to provide an insight into the function of DAP during pregnancy. Parallel studies were performed on rat uteri during the oestrous cycle and oil-induced deciduomata. DAP transcripts were absent in virgin uteri and during the day of implantation (day 5), low in early gestation tissues (days 8, 9) and high during midgestation (days 10–14). Expression of mRNA encoding DAP was first detected on day 8 of pregnancy in decidual cells of the lateral decidual glycogen wing areas. On day 9, mRNA encoding DAP was found in the same area as on day 8 as well as in the outer layer of cells in the antimesometrial decidua adjacent to the myometrium termed the fibrinoid capsula. On days 11 and 12, expression appeared in the decidual cells of the mesometrial decidua, and was particularly strong around the ectoplacental cone and blood vessels that were invaded by the trophoblast. By day 13 and throughout the remainder of pregnancy, expression occurred in the mesometrial decidua and metrial gland. No signal was observed in granulated metrial gland cells. mRNA encoding DAP was also found in the smooth circular muscle coat adjacent to the mesometrial decidua. A similar pattern and cellular localization of expression of mRNA encoding DAP expression was detected during the development of the deciduomata, artificially induced in the absence of a conceptus. The spatial and temporal patterns of expression correlated with a possible role for DAP in mediating fetal–maternal interactions and the establishment of the placental structure.
S. C. Bell, S. R. Patel, P. H. Kirwan and J. O. Drife
Summary. To identify markers of endometrial differentiation specimens of endometrium from the menstrual cycle were incubated in vitro with [35S]methionine, in the absence or presence of progesterone, and protein synthesis and secretion were studied by fluorographic analysis of one dimensional SDS/gradient polyacrylamide gels. Changes were demonstrated in the rate of synthesis and secretion of a number of endometrial proteins (EP) during the cycle and in response to progesterone. Endometrial proteins were classified into three groups: Group I—synthesized and secreted throughout the menstrual cycle and unaffected by progesterone exposure; Group II—synthesis and secretion associated with histological type of endometrium and unaffected by progesterone exposure, e.g. EP 13 (M r 33 000) with proliferative, EP 15 (M r 28 000) with secretory and EP 14 (M r 32 000) with late secretory endometrium; Group III—synthesis and secretion regulated by progesterone exposure irrespective of source of endometrium, e.g. EP 9 (M r 54 000) and 11 (M r45 000). The Group II proteins EP 14 and 15 were also the major secretory protein products of endometrium from first and second trimester pregnancy respectively, the native forms referred to as pregnancy-associated endometrial α1- and α2-globulins (α1- and α2-PEG). We conclude that EP 15 (α2-PEG) represents a human analogue of uteroglobin.