Differential alterations in the distribution of three phosphatase enzymes during the plasma membrane transformation of uterine epithelial cells in the rat.

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of three phosphatase enzymes: 5'-nucleotidase (5N); thiamine pyrophosphatase (TPP); and adenosine triphosphatase (ATP) in the rat endometrium during early pregnancy up to the time of blastocyst attachment. The authors were particularly interested in changes in the apical plasma membrane and reaction product for all three enzymes was clearly localized along this membrane especially on day 1 of pregnancy. However, the three enzymes showed markedly different patterns of organization of reaction product at later times during early pregnancy. 5N, while showing a continuous lining along the microvilli on day 1 was virtually undetectable by day 6. TPP was also strongly present apically on day 1, but reaction product was not always found as a continuous lining. Again, by day 6, there was no presence of this enzyme along the apical surface. ATP differed from the other two in that it produced a strong, and relatively unchanged reaction product along the apical plasma membrane from day 1 through to day 6 of pregnancy. The changes in distribution of these enzymes was particularly obvious at the electron microscopic level and we consider their contribution to the process of 'plasma membrane transformation' of early pregnancy.     

Changes in rat luteal ultrastructure and P450scc mRNA and protein content after in vivo treatment with a gonadotropin-releasing hormone agonist

Previous studies have demonstrated that plasma progesterone levels decrease in pregnant rats treated in vivo with a gonadotropin-releasing hormone agonist (GnRH-Ag), without changes in testosterone or estradiol levels in ovarian vein plasma. The objective of this study was to determine the loci of GnRH-Ag disruption of progesterone synthesis by examining luteal mitochondria, lipid droplets, cellular composition, and P450 side-chain cleavage (P450scc) enzyme and mRNA content in the pregnant rat. On Day 7 or 11 of pregnancy, osmotic minipumps containing GnRH-Ag were implanted into 5-7 rats. Sham operations were performed on 5-6 controls at each time period. Five micrograms per day of GnRH-Ag were released for about 24 h, after which corpora lutea and jugular vein plasma were collected. The corpora lutea were prepared for microscopy or analyzed for P450scc enzyme and mRNA content. Plasma progesterone levels were measured by RIA. In those rats treated with GnRH-Ag, progesterone levels had decreased, and within the luteal cells, there was an increase in the number of lipid droplets and a decrease in the number of tubular cristae within the mitochondria. Concomitantly, P450scc enzyme and mRNA content decreased on both Day 8 and Day 12 of pregnancy. Also, GnRH-Ag treatment decreased the ratio of large to small steroidogenic luteal cells on Day 8 of pregnancy, but did not alter cellular ratios on Day 12 of pregnancy. These observations suggest that treatment with GnRH-Ag inhibits progesterone synthesis by decreasing the amount of P450scc mRNA and enzyme content, which may alter the mitochondrial cristae structure on Day 8 and Day 12 of pregnancy. The reduction in tubular cristae and P450scc enzyme in the mitochondria may account for the increase in lipid droplets, as less cholesterol is converted to pregnenolone. An additional mechanism of inhibition may be the reduction in the number of large steroidogenic luteal cells, which appear to be the major source of progesterone in the rat corpus luteum on Day 8 of pregnancy.     

Increase in lysosomal numbers and activity in the rat uterine luminal and glandular epithelium during early pregnancy: a histochemical study

Lysosomal acid phosphatase was studied at both the light and electron microscope level in the rat uterine luminal and glandular epithelium, at oestrous, late dioestrous and day 6 of pregnancy. At the light-microscopic level lysosomal numbers were quantified and statistically analysed. A morphological study was also carried out on the lysosomes at the electron-microscopic level in the above-mentioned stages. Quantification of lysosomal numbers found day 6 of pregnancy to have a significantly higher lysosomal population in the luminal and glandular epithelium compared to non-pregnant states. At the electron-microscopic level the luminal epithelial lysosomes were frequently observed in an invaginated or vesiculated form whereas these characteristics were rarely observed during late dioestrous and were non-existent during oestrous. Generally, the lysosomes appeared more active in the luminal epithelium at day 6 of pregnancy compared to the non-pregnant state. The findings are discussed in reference to the role of the lysosome at the time of blastocyst implantation.     

Production of slow alpha2-globulin in the pregnant rat.

Slow alpha2-globulin (salpha2G), a high molecular weight glycoprotein, appears as a component of rat serum in a wide variety of both physiologic and pathologic conditions, including pregnancy; it is also present in the neonate. In the present study, the protein was detectable in serum after 6 to 10 days, and was present at moderate levels on day 13, and thereafter through the rest of pregnancy. From immunofluorescent localization and 14C-labelled amino acid incorporation studies, salpha2G was found to be localized in uterine and placental tissues and to be synthesized by these tissues as early as day 6 of gestation. Production continued throughout pregnancy. Synthesis in the liver of the pregnant rat began at day 17 of gestation, which is in contrast with the observation in pathologic conditions that liver synthesis is an initial source of the serum protein; substantial fetal liver synthesis was occurring at day 21 of gestation, and the amount in fetal serum was four times greater than that in maternal serum at that time. It is likely that the fetal liver produces salpha2G as soon as it begins to function.     

Glycosaminoglycans of the aorta in avitaminosis]

It has been shown that pregnancy specific beta 1-globulin (SBG) is found in the rat blood serum on the 6--7th day of pregnancy. SBG reaches the maximum by the end of pregnancy and is not detected already at the 3d--4th day after delivery. The placenta is the site of the SBG snythesis. Other organs of the pregnant rat are not able to incorporate radioactive aminoacids into the protein in vitro.     

Heparin-binding EGF-like growth factor is seen on the extracellular surface of uterine epithelial cells only after the initial stages of blastocyst attachment

The presence and distribution of heparin-binding epidermal growth factor in rat uterine epithelial cells was determined immunohistochemically and localized ultrastructurally. Rat uterine tissue was examined on days 1, 3, 6 and 8 of pregnancy and it was found that while presence of this growth factor was evident from day 1, spatial reorganization occurred by the time of blastocyst implantation. Strong apical staining was evident from day 6 to day 8, day 6 being the approximate time of blastocyst implantation. Electron microscopy further revealed that this growth factor while shown to be expressed very strongly apically from day 6, actually localized on the plasma membrane only after attachment of the blastocyst. This suggests that heparin-binding epidermal growth factor is not involved in the initial stages of implantation but is more likely involved in the post attachment stages of pregnancy.     

Alkaline phosphatase distribution in the plasma membrane of uterine epithelial cells is markedly altered during early pregnancy in the rat

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of alkaline phospatase in the apical plasma membrane of rat uterine epithelial cells during different stages of early pregnancy up to the time of attachment of the blastocyst. Reaction product generated by alkaline phosphatase (AP) was located along the apical plasma membrane at each stage investigated. However, a very different organization of reaction product was observed depending on the time during early pregnancy with a continuous pattern appearing all along the microvilli on day 1. This pattern was subsequently converted into a clumped and highly ‘patchy’ appearance around the time of blastocyst attachment by day 6 of pregnancy. This change in pattern and distribution was only seen on the luminal epithelial cells with glandular epithelial cells and blood vessels displaying an unchanging distribution.     

The uptake of [3H]uridine into the nucleic acids of rat uterus during early pregnancy

1. Changes in content and uptake of [(3)H]uridine into the nucleic acids of rat uterus during the first 9 days of pregnancy were studied. 2. From day 6 implantation sites were separated from the rest of the uterine tissue for independent analysis. 3. Up to day 5 of pregnancy no changes were found in the total dry matter or in RNA and DNA content/unit dry matter nor in the RNA/DNA ratios. 4. From day 6, when implantation sites are visible, the water content of the implantation sites increased by 2-3%, and the RNA content/unit dry wt. and the RNA/DNA ratios increased. The DNA content/unit dry wt. did not increase in the implantation sites until day 8. 5. Uptake of [(3)H]uridine into the acid-soluble fraction of the tissues was markedly higher in implantation sites than in non-implantation sites. 6. Uptake of [(3)H]uridine into RNA was significantly increased on day 3 of pregnancy and again on day 5. 7. On days 6 and 7, the incorporation into RNA of implantation sites was significantly higher than in the remainder of the uterine tissue but decreased on days 8 and 9 to the same value as that of the normal tissue. 8. No change occurred in uptake into DNA until day 6, when there was an increase in uptake by the implantation sites. 9. It is suggested that the increase in RNA synthesis on day 3 is a preparation of the uterus for the onset of implantation on day 5, and that increased synthesis in implantation sites on days 6 and 7 is the elaboration of new RNA necessary for this early stage of pregnancy to commence.     

Trypanosoma lewisi: termination of pregnancy in the infected rat

Trypanosoma lewisi has previously been described as a nonpathogenic parasite of the rat, but these experiments demonstrate that both embryonal and maternal death may occur in the pregnant rat after infection with this parasite.Rats infected early in the first week of pregnancy resorbed their young with little apparent difficulty, and exhibited parasitemia curves typical of nonpregnant infected females of similar age.Rats infected late in the first week of pregnancy experienced greater difficulty resorbing the young, with half of the females dying shortly before parturition. The parasitemia counts were also similar to those of nonpregnant infected rats.The majority of rats infected during the midterm of pregnancy died at the time of parturition, without giving birth to their young. The number of parasites in these animals was abnormally high compared to nonpregnant infected females. Unusually large numbers of dividing trypanosomes were present in the placentae of these animals, many of them containing 8–16 nuclei and kinetoplasts.Animals infected during the last week of pregnancy gave birth to litters of normal size with little apparent difficulty, and had extremely low parasite counts.The hematocrits of all groups of infected animals showed a decrease at the time of peak parasitemia, and the hematocrits of all groups of pregnant rats showed a decrease at the time of birth, except for those infected when day 2 pregnant. These animals completely resorbed their young. The weight losses of rats infected on day 2 and day 6 of pregnancy reflected a termination of pregnancy.     

Enzymic activity in rat uterus during early pregnancy.

Total protein, RNA and DNA content and the activity of acid and alkaline phosphatases, 5'-nucleotidase and isocitrate dehydrogenase were studied in rat uterus during the first 8 days of pregnancy. Isocitrate dehydrogenase activity showed marked fluctuations from day to day. Nucleotidase and acid phosphatase activities showed a significant increase on day 8. The most marked change in activity was that of alkaline phosphatase which showed a 10-fold increase between days 6 and 8, due largely to an increase in the activity of this enzyme in the decidual nodule. The rise in alkaline phosphatase activity did not occur in rats ovariectomized on days 1, 2 or 4 of pregnancy and was markedly decreased in those ovariectomized on day 6. [3H]-uridine incorporation into RNA showed a significant increase between days 2 and 6 whereas [3H]-thymidine incorporation into DNA showed a significant increase on day 6.     

Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only 1/20 of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.     

Desmosomes in uterine epithelial cells decrease at the time of implantation: an ultrastructural and morphometric study

Displacement of uterine epithelial cells is an important aspect of implantation in the rat and other species, allowing invasion of the blastocyst into the endometrial stroma. Desmosomes, which are part of the lateral junctional complex, function in cell-to-cell adhesion, and are therefore likely to be involved in displacement of uterine epithelial cells at the time of implantation. This study used transmission electron microscopy to study rat uterine epithelial cells during the peri-implantation period to investigate the change in the number of structural desmosomes along the lateral plasma membrane of uterine epithelial cells. We found a significant decrease in the number of desmosomes along the entire lateral plasma membrane as pregnancy progressed. Furthermore, there were also significant decreases in the number of desmosomes on the apical portion of the lateral plasma membrane between all days of pregnancy examined. In addition, on day 6 of pregnancy, the time of attachment, desmosomes were larger and seen as "giant desmosomes." For the first time, this study has shown that there is a significant reduction in cell height and actual number of ultrastructurally observable desmosomes at the time of implantation in the rat. It is proposed that this reduction in desmosome number leads to a decrease in lateral adhesion between uterine epithelial cells at the time of implantation, and hence is involved in the loss of uterine epithelial cells to facilitate blastocyst invasion.     

Expression of steroidogenic acute regulatory protein mRNA in rat placenta during mid-late pregnancy

The production of a steroid hormone in the placenta is essential for maintaining the pregnancy and developing the fetus during gestation. In various steroidogenic tissues (including gonads and adrenal cortex), the steroidogenic-acute-regulatory protein (StAR) acutely transfers cholesterol from the outer to the inner mitochondrial membrane for rapid steroidogenesis. Although steroid hormones were synthesized in the rat placenta, the developmental expression of StAR has been poorly understood in the rat placenta during mid-late pregnancy. Therefore, the aim of the present study was to establish the expression and localization of StAR mRNA in the rat placenta during mid-late pregnancy using Northern blots and in situ hybridization. The Northern blot analysis showed that the StAR mRNA expression significantly changed as the gestation day (GD) progressed. The placental expression of StAR mRNA increased between GD 11 and 13, and then slightly decreased until term. In situ hybridization showed a strong StAR expression in giant trophoblast cells on GD 11 and 13, and a moderate expression in trophoblast and stroma cells within the villi of the labyrinth zone throughout the pregnancy. In this study, we reveal for the first time the existence of StAR mRNA in steroidogenic cells of the placenta during mid-late pregnancy. In conclusion, our results suggest that StAR may regulate steroidogenesis in the rat placenta to maintain the pregnancy and developing the fetus.     

Cyclic nucleotides and lipids in the realization of the radioprotective effect of ceruloplasmin

Ceruloplasmin administered 60 min before irradiation diminished cAMP and cGMP levels, which were increased by irradiation at LD50 and LD100, and normalized cAMP/cGMP ratio in the rat liver during the first 24 h following irradiation with a dose of 6.24 Gy. The content of phospholipids increased and that of cholesterol decreased under the effect of ceruloplasmin leading to normalization of the molar cholesterol/phospholipid ratio in the rat liver on the 7th day of radiation sickness (LD50, 6.24 Gy).     

ICAM1 and fibrinogen-γ are increased in uterine epithelial cells at the time of implantation in rats

Uterine epithelial cells transform into a receptive state to adhere to an implanting blastocyst. Part of this transformation includes the apical concentration of cell adhesion molecules at the time of implantation. This study, for the first time, investigates the expression of ICAM1 and fibrinogen‐γ (FGG) in uterine epithelial cells during normal pregnancy, pseudopregnancy and in hormone‐treated rats. An increase (P < 0.05) in ICAM1 was seen at the apical membrane of uterine epithelial cells at the time of implantation compared with day 1 of pregnancy. ICAM1 was also increased (P < 0.05) on day 6 of pseudopregnancy as well as in ovariectomized rats treated with progesterone plus oestrogen. These results show that ICAM1 up‐regulation at the time of implantation is under the control of progesterone, and is not dependent on cytokine release from the blastocyst or in semen. FGG dimerization increased (P < 0.05) on day 6 of pregnancy compared with day 1, and was not up‐regulated in day 6 pseudopregnant animals, suggesting this increase is dependent on a developing blastocyst. The presence of ICAM1 and FGG in the uterine epithelium at the time of implantation in the rat is similar to that seen in lymphocyte–endothelium adhesion, and we suggest a similar mechanism in embryo–uterine epithelium adhesion is utilized. Mol. Reprod. Dev. 78:318–327, 2011. © 2011 Wiley‐Liss, Inc.     

Alterations in uterine and placental prostaglandin F and E with gestational age in the rat

Experiments were designed to determine the chronological alterations in placental and uterine prostaglandin F and E (PGF and PGE) during pregnancy in the rat. Pregnant rats (sperm in the vagina = day 0) were sacrified at days 15, 18,19, 20, 21 and delivery (day 21 ) and placental and uterine tissues assayed (RIA) for PGF and PGE immediately (“ ”) or after 1 hour incubation (“ ”). Uterine content of PGF and PGE (ng PG/mg DNA) was increased significantly by day 19 and further increases were seen through delivery. Incubation of uterine tissue resulted in enhanced net production of PGF and PGE (p <.05) per mg DNA (as judged by tissue content and release into the incubation medium) by day 18 of pregnancy vs. day 15. Net production peaked around the time of delivery thus paralleling the alterations in tissue content .By contrast, no differences with gestational age were found in placental content of PGF and PGE , the concentrations throughout late gestation remaining in the range of uterine PGs at day 15. However, production of PGs per mg placental DNA increased markedly during incubation with significant enhancement detected by day 19 vs. 15, achieving levels even greater than the uterus .The and findings for the uterus are consistent with the hypothesis that increases in uterine PGs levels at the end of pregnancy may play an important role in parturition. The experiences with placental tissue suggest that the potential for PG production per placental cell may also increase in late gestation and thereby contribute to the augmented intrauterine availability of PGs at that time.     

Alteration of GABA levels in ovary and fallopian tube of the pregnant rat

The concentration and the total quantity of gamma-aminobutyric acid (GABA) were determined in ovary and Fallopian tube of the rat on day 3, 7, 14 and 21 of pregnancy and the values were compared with a diestrous control group. In ovary, both GABA concentration and total GABA content gradually elevated up to day 14 of gestation but, thereafter, the diminution of the two parameters was observed. The GABA concentrations in Fallopian tube were found to be strongly reduced during pregnancy, and the total amount of the amino acid in this organ also decreased markedly. The significant alterations of GABA levels in both organs suggest a possible correlation between the functional status and GABA content of ovary and Fallopian tube of the pregnant rat.