Eyelid pigment implantation: early and late histopathology

Full-thickness sections of upper and lower eyelids were obtained from patients who had eyelid pigment implantation performed by one surgeon. These four patients represented eyelid pigment implantation at varying postoperative stages ranging from 6 months to 4 years. Light microscopic evaluation revealed pigment within dermis and superficial orbicularis muscle. Light and electron microscopic evaluation revealed the vast majority of the pigment to be located intracellularly, primarily within macrophages. No foreign-body reaction was seen around the implanted material. Electron probe analysis of the pigment showed only the presence of iron. Analysis of the unused pigment revealed small amounts of silica and magnesium. These substances could not be identified by electron probe analysis in the eyelid tissues.     

Cell subpopulations in the late morula and early blastocyst of the mouse

Late morulae and early blastocysts consist of two main cell subpopulations which occupy different positions within the embryos. The cells of the outer layer have a polar surface phenotype. The outward-facing surface of this cell type has a discrete dense pole of short microvilli, whilst the inward-facing surface has a relatively sparse distribution of longer, thick microvilli. The inner cells lack short, dense microvilli but exhibit thick microvilli of variable density. After short-term isolation in medium low in Ca²⁺, the individual polar and apolar cells remain distinguishable. The expanded blastocyst also has two major cell subpopulations, but within each of these, heterogeneity is observed. The mural trophectodermal cells have a larger, more regular outward-facing area of sparse, short microvilli than do polar trophectodermal cells. The ICM consists of some cells that show extensive blebbing in medium low in Ca²⁺ and others that do not.     

Requirement for progesterone priming and its long-term effects on implantation in the mouse

At least 48 hr of progesterone (P4) priming has been documented to be essential for P4 and estrogen to initiate implantation in the rat. However, the length of this P4 priming requirement for implantation in the mouse has not been experimentally defined. Therefore, our first objective was to determine the length of P4-priming requirement for implantation in the mouse. Day 4 blastocysts were transferred into the uteri of Day 5 or Day 6 pseudopregnant mice that were ovariectomized on Day 1 (= vaginal plug) and treated with a single injection of P4 and 17 beta-estradiol (E2) only on Day 5, or a single injection of P4 on Day 5 followed by a second injection of P4 plus E2 on Day 6, respectively. Although none of the transferred blastocysts implanted in the uteri of P4-unprimed recipients, 46% of the transferred blastocysts implanted into the uteri of all recipients that were first primed with P4 24 hour prior to a second injection of P4 and E2. These results suggest that in contrast to the rat, the mouse uterus requires at most 24 hr of P4 priming before P4 and estrogen can initiate implantation. Our second objective was to determine whether P4 priming has a long-term effect on implantation in the mouse. Our present results and those of others suggest that the mouse uterus is exposed to rising P4 levels for 24 hr prior to implantation on Day 4 of pregnancy. Therefore, in the present investigation, induction of implantation by an injection of P4 and E2 following 5 days of ovariectomy performed on Day 4 of pregnancy clearly suggests that once exposed to P4 for 24 hr, the mouse uterus retains a long-term effect, i.e., following P4 withdrawal for several days, 24 hr of initial P4 priming is no longer required for P4 and estrogen to initiate implantation. Our next objective was to explore whether this long-term effect of P4 priming on implantation can be prolonged and potentiated by increasing the length of initial P4 priming. Thus, when the mice were ovariectomized on Day 4 of pregnancy and treated with P4 beginning on Day 5 for 4 days, the long-term effect on implantation was prolonged (8 days vs 5 days following P4 withdrawal) and potentiated (94% vs 0% mice with implantation following 8 days of P4 withdrawal) as compared with those with no P4 priming after ovariectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocysts. The enzyme activities are distinguishable by means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-l-lysine-chloromethyl ketone or l-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

Possible effects of pituitary adenylate cyclase activating polypeptide (PACAP) on early embryo implantation marker HB-EGF in mouse

Pituitary adenylate cyclase activating polypeptide (PACAP) was originally isolated as a hypothalamic neuropeptide stimulating adenylate cyclase activity. Besides its neuroprotective effects, numerous data proved its role in reproductive processes. However, there are limited data on its role in preimplantation embryo development and implantation. Our aim was to analyse the mRNA expression of Adcyap1 (coding region of PACAP) and Hbegf [coding region of HB-EGF (Heparin-binding EGF-like growth factor)] in embryos and pregnant uterus to investigate the possible correlation between them. Eight-week-old BDF1 mice were superovulated and subsequently mated overnight or left in their cage after hCG treatment. Day4 embryos were flushed from mated females. After morphological analysis, Adcyap1 and Hbegf gene expression of embryos and uterine tissues was assessed with qPCR.Our results showed significantly higher Adcyap1 and Hbegf mRNA levels in females producing embryos compared to non-mated ones. Robust elevation of Adcyap1 and slight elevation of Hbegf were detected in females with blastocyst embryos compared with non-blastocysts. We found low rate of Hbegf mRNA expression in uncompacted embryos, whereas morulae and blastocysts expressed high amounts of Hbegf. However, we did not find detectable Adcyap1 mRNA in embryos. Strong correlation was found between uterine tissue and embryonic Hbegf levels, slight correlation between uterine Adcyap1 and Hbegf levels. Uterine tissue Adcyap1 and embryonic Hbegf showed no correlation. In summary, our present data show, for the first time, the correlation between PACAP and HB-EGF mRNA expression suggesting that PACAP might play a role during the peri-implantation period of early mouse embryo development.     

Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts.

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocyts. The enzyme activities are distinguishable my means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-L-lysine-chloromethyl ketone or L-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.     

Developmental abnormalities of NT mouse embryos appear early after implantation

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.     

Expression of maspin in the early pregnant mouse endometrium and its role during embryonic implantation

Maspin (serpin B5), a tumor-suppressing member of the serine protease inhibitor family, participates in cell migration, adhesion, invasion, and apoptosis. These processes are also critical for embryo implantation, but the role of maspin in embryo implantation remains poorly understood. The aim of the present study was to investigate the spatiotemporal expression of maspin in early pregnant mouse endometrium and its role in embryo implantation. Real-time quantitative PCR analysis, immunohistochemistry, and Western blotting were used to detect mRNA and protein expression of maspin in the endometria of nonpregnant and early pregnant (days 0 to 7) mice. On day 3 of pregnancy, mice in the treated group (n = 20) were injected in the left uterine horn with antimaspin polyclonal antibody and in the right horn with purified rabbit IgG; control mice (n = 20) were injected only with purified rabbit IgG in the right uterine horn. Implanted embryos were counted on pregnant day 8. The mRNA and protein expressions of maspin were higher in the endometria of pregnant mice than nonpregnant mice; these levels gradually increased from day 1 of pregnancy, peaked on day 5, and then decreased on days 6 and 7. The mice treated with antimaspin polyclonal antibody group had far fewer implanted embryos than did the control group. Taken together, these results suggest that maspin, a tumor suppressor, may play an important role in embryo implantation.     

Priority effects of the early breeding fire salamander on the late breeding banded newt

Early breeding intraguild predators may have advantages over late breeding predators via priority effects; early breeding predators may reduce shared prey resources before late breeders appear and may also prey upon the late breeders. Here we show that predatory larvae of the late-breeding predatory banded newt, Triturus vittatus vittatus, occupy the same temporary pond toward the end of the developmental period of the early-breeding predatory fire salamander, Salamandra salamandra, resulting in a large size disparity between larvae of these two species while they co-occur. We conducted outdoor artificial pool experiments to assess priority effects of large larval Salamandra at the end of their larval development period, on recently hatched larval Triturus. We also assessed how artificial vegetation may influence larval Triturus performance in the presence or absence of Salamandra Salamandra, introduced into the experimental pools two weeks prior to the newt larvae, strongly reduced invertebrate prey abundance shared by these two predatory urodeles and with only a one week period of overlap, strongly reduced abundance of Triturus larvae. The artificial vegetation had only a small ameliorating effect on Triturus survival when Salamandra was present. Triturus size at metamorphosis (snout-tail length) was significantly larger in the Salamandra pools, presumably due to a combination of a strong “thinning effect” and greater vulnerability of smaller Triturus individuals to predation by Salamandra. Time to metamorphosis was not significantly affected by Salamandra. These results have conservation implications as T. v. vittatus is listed as highly endangered and may also explain the largely negative spatial association of the two species. Handling editor: K. Martens     

Contrasting effects of early and late orchiectomy on hypertension and renal disease in fawn-hooded rats

Fawn-hooded (FH) rats, primarily males, develop spontaneous low-renin hypertension associated with reduced urinary excretion of kallikrein as early as 2 months of age, followed by progressive glomerular sclerosis and proteinuria as early as 3 months of age. In the present study we determined the effects of early (5-7 weeks) or late (5 months) orchiectomy on the blood pressure and nephropathy of FH rats, compared to sham-operated (control) FH males. Early orchiectomy reduced significantly the progression of glomerular sclerosis and of proteinuria and ameliorated the hypertension but had no significant effect on excretion of urinary kallikrein. Late orchiectomy, in contrast, had no significant effect on the progression of glomerular sclerosis or proteinuria but did significantly reduce the blood pressure and marginally increase the excretion of urine kallikrein. These results suggest that (a) male sex hormones may play a role in the pathogenesis of hypertension and nephropathy in the FH rats and (b) renal disease in this strain progresses in spite of improvement in blood pressure.     

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis

 The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20–80 μM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process. Accepted: 13 January 1999