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Shark placentae are derived from modifications to the fetal yolk sac and the maternal uterine mucosa. In almost all placental sharks, embryonic development occurs in an egg capsule that remains intact for the entire pregnancy, separating the fetal tissues from the maternal tissues at the placental interface. Here, we investigate the structure and permeability of the egg capsules that surround developing embryos of the placental Australian sharpnose shark (Rhizoprionodon taylori) during late pregnancy. The egg capsule is an acellular fibrous structure that is 0.42 ± 0.04 μm thick at the placental interface between the yolk sac and uterine tissues, and 0.67 ± 0.08 μm thick in the paraplacental regions. This is the thinnest egg capsule of any placental shark measured so far, which may increase the diffusion rate of respiratory gases, fetal wastes, water and nutrients between maternal and fetal tissues. Molecules smaller than or equal to ~ 1000 Da can diffuse through the egg capsule, but larger proteins (~ 3000–26,000 Da) cannot. Similar permeability characteristics between the egg capsule of R. taylori and other placental sharks suggest that molecular size is an important determinant of the molecules that can be exchanged between the mother and her embryos during pregnancy.

Introduction Embryonic growth and development require efficient respiratory gas exchange. Internal incubation of developing young thus presents a significant physiological challenge, because respiratory gas diffusion to embryos is impeded by the additional barrier of parental tissue between the embryo and the environment. Therefore, live-bearing species exhibit a variety of adaptations facilitating respiratory gas exchange between the parent (usually the mother) and embryos. Syngnathid fishes are the only vertebrates to exhibit male pregnancy, allowing comparative studies of the biology and evolution of internal incubation of embryos, independent of the female reproductive tract. Here, we examine the fleshy, sealed, seahorse brood pouch, and provide the first quantification of structural changes to this gestational organ across pregnancy. Methods We used histological analysis and morphometrics to quantify the surface area for exchange across the brood pouch epithelium, and the structure of the vascular bed of the brood pouch. Results We show dramatic remodelling of gestational tissues as pregnancy progresses, including an increase in tortuosity of the gestational epithelium, an increase in capillary density, and a decrease in diffusion distance between capillaries and the pouch lumen. Discussion These changes produce an increased surface area and expansion of the vascular bed of the placenta that likely facilitates respiratory gas exchange. These changes mirror the remodelling of gestational tissue in viviparous amniotes and elasmobranchs, and provide further evidence of the convergence of adaptations to support pregnancy in live-bearing animals.

There are many different forms of nutrient provision in viviparous (live-bearing) species. The formation of a placenta is one method where the placenta functions to transfer nutrients from mother to fetus (placentotrophy), to transfer waste from the fetus to the mother, and to perform respiratory gas exchange. Despite having the same overarching function, there are different types of placentation within placentotrophic vertebrates, and many morphological changes occur in the uterus during pregnancy to facilitate formation of the placenta. These changes are regulated in complex ways but are controlled by similar hormonal mechanisms across species. This review describes current knowledge of the morphological and molecular changes to the uterine epithelium preceding implantation among mammals. Our aim is to identify the commonalities and constraints of these cellular changes to understand the evolution of placentation in mammals and to propose directions for future research. We compare and discuss the complex modifications to the ultrastructure of uterine epithelial cells (UEC) and show that there are similarities in the changes to the cytoskeleton and gross morphology of the UEC, especially of the apical and lateral plasma membrane of the cells during the formation of a placenta in all eutherians and marsupials studied to date. We conclude that further research is needed to understand the evolution of placentation among viviparous mammals, particularly concerning the level of placental invasiveness, hormonal control, and genetic underpinnings of pregnancy in marsupial taxa.

Porosomes are plasma membrane structures in secretory cells that allow transient docking and/or partial fusion of vesicles during which they release their content then disengage. This is referred to as "kiss and run" exocytosis. During early pregnancy, at the time of receptivity, there is a high level of vesicle activity in uterine epithelial cells (UECs). One of the secretory pathways for these vesicles could be via porosomes, which have yet to be identified in UECs. This study identified porosomes in the apical plasma membrane of UECs for the first time. These structures were present on days 1, 5.5, and 6 of early pregnancy, where they likely facilitate partial secretion via "kiss and run" exocytosis. The porosomes were measured and quantified on days 1, 5.5, and 6, which showed there are significantly more porosomes on day 5.5 (receptive) compared to day 1 (nonreceptive) of pregnancy. This increase in porosome numbers may reflect major morphological and molecular changes in the apical plasma membrane at this time such as increased cholesterol and soluble NSF attachment protein receptor proteins, as these are structural and functional components of the porosome complex assembly. Porosomes were observed in both resting (inactive) and dilated (active) states on days 1, 5.5, and 6 of early pregnancy. Porosomes on day 5.5 are significantly more active than on day 1 as demonstrated by the dilation of their base diameter. Further two-way ANOVA analysis of base diameter in resting and dilated states found a significant increase in porosome activity in day 5.5 compared to day 1. This study therefore indicates an increase in the number and activity of porosomes at the time of uterine receptivity in the rat, revealing a mechanism by which the UECs modify the uterine luminal environment at this time.

Introduction Viviparity (live-birth) has evolved from oviparity (egg-laying) multiple times in sharks. While most transitions from oviparity to viviparity have resulted in non-placental forms of viviparity, some sharks develop a yolk sac placenta during pregnancy. The Australian sharpnose shark (Rhizoprionodon taylori) is a placental species that suspends embryonic development in a diapause for most of pregnancy. Methods To identify structures involved in supporting rapid embryonic growth in late pregnancy, we examined uterine and placental morphology by light and electron microscopy. Results Paraplacental uterine regions have morphological specialisations consistent with secretion and fluid transport between uterine tissues and the lumen. Uterine secretions in the lumen may be absorbed by the outgrowths on the embryonic umbilical cord (‘appendiculae’), which are densely covered by microvilli. The placenta consists of uterine villi that interdigitate with the yolk sac and enhance the surface area available for fetomaternal exchange. The yolk sac does not invade the uterine epithelium, and the egg capsule remains intact at the placental interface, separating maternal and fetal tissues. Some placental uterine epithelial cells are secretory, and endocytic vesicles in the opposing yolk sac ectodermal cells suggest that nutrient transport is by histotrophic uterine secretion followed by fetal absorption. Respiratory gases, water and possibly small nutrients likely diffuse across the placenta, where maternal and fetal blood vessels are ~2 μm apart. Discussion Placental structure in R. taylori is similar to most other sharks, but there are differences in cellular structures between species that may indicate species-specific placental transport mechanisms.

The luminal uterine epithelial cells are the first point of contact with the implanting blastocyst. Dramatic changes occur in the structure and function of these cells at the time of receptivity including changes in the lateral junctional complex. While these morphological changes are important for uterine receptivity, currently there is no known mechanism of regulation of the lateral junctional complexes. Rab13, a member of the Rab (Ras-related in the brain) family of GTPases has a critical role in endosomal trafficking to the lateral plasma membrane and is involved in modulation of the tight junction in several cell types. The aim of this study is to investigate the role of Rab13 in changes to the lateral junctional complex at the time of receptivity. Immunofluorescence microscopy demonstrated no association between Rab13 and ZO-1 (a tight junction protein) or Rab13 and E-cadherin (an integral component of adherens junctions). Co-localisation was demonstrated between Rab 13 and desmoglein-2 at the time of fertilization and also at receptivity suggesting involvement of Rab13 in relocalisation of desmoglein-2 and formation of giant desmosomes in the apical part of the lateral plasma membrane at the time of uterine receptivity. We suggest that despite the loss of the adherens junction at the time of receptivity, the presently reported redistribution of desmosomes regulated by Rab13 allows the uterine epithelium to maintain structural integrity.

Embryos of the viviparous dwarf ornate wobbegong shark (Orectolobus ornatus) develop without a placenta, unattached to the uterine wall of their mother. Here, we present the first light microscopy study of the uterus of O. ornatus throughout pregnancy. At the beginning of pregnancy, the uterine luminal epithelium and underlying connective tissue become folded to form uterine ridges. By mid to late pregnancy, the luminal surface is extensively folded and long luminal uterine villi are abundant. Compared to the nonpregnant uterus, uterine vasculature is increased during pregnancy. Additionally, as pregnancy progresses the uterine epithelium is attenuated so that there is minimal uterine tissue separating large maternal blood vessels from the fluid that surrounds developing embryos. We conclude that the uterus of O. ornatus undergoes an extensive morphological transformation during pregnancy. These uterine modifications likely support developing embryos via embryonic respiratory gas exchange, waste removal, water balance, and mineral transfer.

The fluid that surrounds the embryo in the uterus contains important nourishing factors and secretions. To maintain the distinct microenvironment in the uterine lumen, the tight junctions between uterine epithelial cells are remodeled to decrease paracellular movement of molecules and solutes. Modifications to tight junctions between uterine epithelial cells is a common feature of pregnancy in eutherian mammals, regardless of placental type. Here we used immunofluorescence microscopy and western blot analysis to describe distributional changes to tight junctional proteins, claudin-1, -3, -4, and -5, in the uterine epithelial cells of a marsupial species, Sminthopsis crassicaudata. Immunofluorescence microscopy revealed claudin-1, -3, and -5 in the tight junctions of the uterine epithelium of S. crassicaudata during pregnancy. These specific claudins are associated with restricting passive movement of fluid between epithelial cells in eutherians. Hence, their function during pregnancy in S. crassicaudata may be to maintain the uterine luminal content surrounding developing embryos. Claudin-4 disappears from all uterine regions of S. crassicaudata at the time of implantation, in contrast with the distribution of this claudin in some eutherian mammals. We conclude that like eutherian mammals, distributional changes to claudins in the uterine epithelial cells of S. crassicaudata are necessary to support pregnancy. However, the combination of individual claudin isoforms in the tight junctions of the uterine epithelium of S. crassicaudata differs from that of eutherian mammals. Our findings suggest that the precise permeability of the paracellular pathway of the uterine epithelium is species-specific.

Luminal uterine epithelial cells (UEC) have a surge in vesicular activity during early uterine receptivity. It has been predicted these vesicles exit the UEC via exocytosis resulting in secretion and membrane trafficking. The present study investigated the changes in SNARE proteins VAMP2 (v-SNARE) and syntaxin 3 (t-SNARE) localisation and abundance in UECs during early pregnancy in the rat. We found VAMP2 and syntaxin 3 are significantly higher on day 5.5 compared to day 1 of pregnancy. On day 5.5, VAMP2 is perinuclear and syntaxin 3 is concentrated in the apical cytoplasm compared to a cytoplasmic localisation on day 1. This change in localisation and abundance show VAMP2 and syntaxin 3 are involved in vesicular movement and membrane trafficking in UECs during early pregnancy. This study also investigated the influence of cytoskeletal disruption of microtubules and actin filaments on VAMP2 and syntaxin 3 in UECs grown in vitro, since microtubules and actin influence vesicle trafficking. As expected, this study found disruption to microtubules with colchicine and actin with cytochalasin D impacted VAMP2 and syntaxin 3 localisation. These results suggest VAMP2 and syntaxin 3 are involved in the timely trafficking of vesicular membranes to the apical surface in UECs during early pregnancy, as are of microtubules and actin.

The uterine surface undergoes significant remodeling, termed the "plasma membrane transformation," during pregnancy to allow for implantation of the blastocyst and formation of the placenta in viviparous amniote vertebrates. Unlike other species within the superorder Euarchontoglires, which have a hemochorial (highly invasive) placenta, kangaroo rats (Dipodomys spp.) exhibit a less invasive endotheliochorial placenta. We characterized the changes that occur to membrane molecules and the cellular ultrastructure of the uterine epithelium during early pregnancy in Merriam's kangaroo rat, Dipodomys merriami using electron microscopy and immunofluorescence microscopy. Epithelial cadherin (E-cadherin) is an adhesion protein that forms the adherens junction and is localized to the lateral plasma membrane of uterine epithelium during the nonreproductive state but localizes nonspecifically in the uterine epithelium immediately preceding implantation. Desmosomes are a type of cadherin that form junctional complexes along the lateral plasma membrane of epithelium. Dsg-2, a marker for desmosomes, is localized along the lateral plasma membrane in non-pregnant animals but redistributes to the apical region of the lateral plasma membrane during early pregnancy. The shift in desmosome and cadherin distribution before implantation suggests that there is a reduction in lateral adhesion between epithelial cells to allow for invasion by the blastocyst. Surprisingly, although Kangaroo rats form a less invasive placenta, these same changes occur during pregnancy in species with highly invasive placentation, such as the laboratory rat and human. These commonalities suggest that it is not through the retention of lateral adhesion that the blastocyst is prevented from further invasion in this rodent species. Anat Rec, 301:1928-1935, 2018. © 2018 Wiley Periodicals, Inc.

Mammals exhibit similar changes in uterine epithelial morphology during early pregnancy despite having a diverse range of placental types. The uterine epithelium undergoes rapid morphological and molecular change ("plasma membrane transformation") during the early stages of pregnancy to allow attachment of the blastocyst. The domestic cat, Felis catus is in the order Carnivora; all species within the Carnivora studied so far develop an endotheliochorial placenta during pregnancy. The endotheliochorial placental type is a common form of placental invasion in mammals. The molecular changes that allow remodeling of the uterine epithelium in preparation for implantation are unknown in most mammals but would provide us with an understanding of what molecules underpin successful implantation and pregnancy among Carnivora. We used immunofluorescence microscopy to localize the key adherens junction proteins desmoglein-2 and E-cadherin in the lateral plasma membrane of the uterine epithelium of F. catus during pregnancy. We show that redistribution of desmoglein-2 and E-cadherin likely facilitates reduction of cell-to-cell adhesion allowing for implantation of the blastocyst and formation of the placenta. The ultrastructural and molecular changes to the uterine epithelium during early pregnancy in F. catus are similar to that in species with other levels of placental invasiveness, suggesting that key molecules such as desmoglein-2 and E-cadherin are crucial to successful pregnancy across all mammals. Anat Rec, 301:1497-1505, 2018. © 2018 Wiley Periodicals, Inc.

For the development of uterine receptivity, many morphological and molecular changes occur in the apical surface of luminal uterine epithelial cells (UECs) including an increase in vesicular activity. Vesicular movements for exocytosis and endocytosis are dependent on microtubules; however, changes in microtubules in UECs during early pregnancy have received little attention. β-tubulin, one of the main component of microtubules, is distributed throughout the cytoplasm of UECs on day 1 (non-receptive) of pregnancy in the rat. On day 5.5, β-tubulin is concentrated above the nuclei and by day 6 (receptive), β-tubulin is concentrated in a band-like fashion above the nucleus. Western blot analysis of isolated UECs found two bands (50 and 34 kDa) for β-tubulin in UECs during early pregnancy. The intensity of the 34 kDa band was significantly higher on day 6 compared to day 1. The increase in the 34 kDa band may be due to higher proteolytic activity associated with microtubule polymerisation during the receptive state. Transmission electron microscopy showed fragmented microtubules at the time of receptivity in UECs. This is the first study to show that microtubules are reorganised during uterine receptivity. This re-organisation likely facilitates vesicular movement and promotes the reorganisation of the apical plasma membrane for uterine receptivity.

Alterations to the basal attachment points between the epithelium of the uterus and the underlying tissue in early pregnancy affect how easily the epithelium can be invaded by the implanting embryo. Attachment points- focal adhesions- disassemble to facilitate highly invasive implantation in rats, but species with less invasive implantation, including marsupials, may require different basal alterations for successful pregnancy. Here we used immunofluorescence microscopy and Western blotting to conduct the first study of basal plasma membrane dynamics in the uterus during marsupial pregnancy. We describe localisation patterns of two key anchoring molecules, talin and paxillin, throughout pregnancy in the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Basal staining of both molecules occurs in early pregnancy, as it does in the rat. However, unlike rats, there is strong basal localisation of talin and paxillin just before implantation in S. crassicaudata, indicating that focal adhesions do not disassemble during pregnancy in this species, and that molecular reinforcement of the epithelium may be a maternal strategy to regulate invasion. Additionally, talin and paxillin do not co-localise at all stages of pregnancy as they do in the rat. Different localisation patterns among mammalian species demonstrate that not all early pregnancy changes are ubiquitous in mammalian pregnancy, as changes to the basal plasma membrane of the epithelium, in particular, may instead be dependent on mode of implantation. Anat Rec, 300:1150-1159, 2017. © 2016 Wiley Periodicals, Inc.

Mammalian pregnancy involves remodelling of the uterine epithelium to enable placentation. In marsupials, such remodelling has probably played a key role in the transition from ancestral invasive placentation to non-invasive placentation. Identifying uterine alterations that are unique to marsupials with non-invasive placentation can thus elucidate mechanisms of marsupial placental evolution. We identified apical alterations to uterine epithelial cells prior to implantation in Monodelphis domestica, a member of the least derived living marsupial clade (Didelphidae) with invasive (endotheliochorial) placentation. We then compared these traits with those of Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae), both with non-invasive placentation, to identify which alterations to the uterine epithelium are ancestral and which facilitate secondarily evolved non-invasive placentation. In M. domestica, remodelling of the uterine epithelium involves reduced cellular heterogeneity and development of uterodome-like cells, suggesting that similar alterations may also have occurred in the marsupial common ancestor. These alterations also overlap with those of both T. vulpecula and Ma. eugenii, suggesting that the placental shift from invasive to non-invasive placentation in marsupials involves essential, conserved characteristics, irrespective of placental mode. However, unique apical alterations of both T. vulpecula and Ma. eugenii, relative to M. domestica, imply that lineage-specific alterations underpin the evolutionary shift to non-invasive placentation in marsupials.

© The Author(s) 2018. The angiogenic factor vascular endothelial growth factor-A (VEGFA) plays a critical role during early pregnancy in many species including the rat, and any alterations in VEGFA levels can severely impact blastocyst implantation rates. The rat ovarian hyperstimulation (OH) model is useful in studying how the induction of superovulation affects VEGFA levels and endometrial receptivity to blastocyst implantation. The present study shows that the major isoform in the rat uterus, Vegf 188 , is reduced at the time of receptivity in OH compared to normal pregnancy, whereas there is no change in Vegf 164 and Vegf 120 messenger RNA (mRNA). The VEGFA receptor 2 (VEGFR2) protein levels are also reduced at the time of receptivity in OH. Our ovariectomy studies show that Vegf 164 , Vegf 188 , and Vegf 120 are significantly decreased by estrogen, and, to a lesser extent progesterone, when compared to control animals. Although no change in the percentage of endometrial blood vessels was seen across all stages of pregnancy, at the time of receptivity in OH pregnancies, blood vessels were typically larger compared to other stages. The altered progesterone–estrogen ratio seen in OH, taken together with our ovariectomy studies, explains the changes to Vegfa mRNA in OH at the time of receptivity. Since VEGFA is important during implantation, the changes to Vegfa and VEGFR2 levels in the endometrium may help explain the observed lower endometrial receptivity following OH. This study aimed to analyse how ovarian hyperstimulation alters the levels of vascular endothleial growth factor and its major receptor, VEGFR2 in the uterus in a rat model.

Controlled ovarian hyperstimulation is an essential component of IVF techniques to ensure proliferation and development of multiple ovarian follicles, but the effects of these hormones on the endometrium are largely unknown. During normal pregnancy in rats, there are significant changes in the basal plasma membrane of uterine epithelial cells (UECs) at the time of receptivity, including loss of focal adhesions. This enables the UECs to be removed from the implantation chamber surrounding the blastocyst, thus allowing invasion into the underlying stroma. This study investigated the influence of ovarian hyperstimulation (OH) on the basal plasma membrane of UECs during early pregnancy in the rat. Immunofluorescence results demonstrate the presence of paxillin, talin, integrin β1 and phosphorylated FAK (Y397FAK) in the basal portion of UECs at the time of implantation in OH pregnancy. TEM analysis demonstrated a flattened basal lamina and the presence of focal adhesions on the basal surface at this time in OH pregnancy. Significantly low full-length paxillin, high paxillin δ and integrin β1 were seen at the time of implantation in OH compared with those in normal pregnancy. The increase in paxillin δ suggests that these cells are less mobile, whereas the increase in integrin β1 and Y397FAK suggests the retention of a stable FA complex. Taken together with the increase in morphological focal adhesions, this represents a cell type that is stable and less easily removed for blastocyst implantation. This may be one mechanism explaining lower implantation rates after fresh embryo transfers compared with frozen cycles.

Uterine luminal epithelial cells (UECs) undergo the plasma membrane transformation in the transition to receptivity. This involves transient alterations in the apical junctional complex (AJC) including increases to the depth and complexity of the tight junction, loss of the adherens junction, and a decrease in the number of desmosomes along the lateral cell membranes. Nectin-3 is key protein involved in the structure and function of the AJC. This study, used immunofluorescence, Western blotting, colocalization, and coimmunoprecipitation analyses, to investigate whether nectin-3 was present in the rat uterus and was regulated by hormones and the blastocyst during early pregnancy. The results showed that nectin-3 was present in UECs as 3 molecular weight protein isoforms (80 kDa, 60 kDa, and 32 kDa). At the time of fertilization (day 1 of pregnancy), nectin-3 was localized basally, but at the time of implantation, (day 6 of pregnancy) when UECs were receptive, nectin-3 increased in the cellular junctions. When UECs returned to the nonreceptive state (day 9 of pregnancy), nectin-3 redistributed back to the cell cytoplasm. This study also showed that nectin-3 localization at the cell junctions was likely to be controlled by progesterone; however, neither ovarian hormones nor the blastocyst regulated protein abundance. This study further showed that while nectin-3 localized to the tight junction at the time of implantation, it did not interact with occludin or l-afadin. These results suggest that at the time of implantation, nectin-3 may contribute to the formation of the tight junction in a protein complex independent from occludin and l-afadin.

Vascular endothelial growth factor A is a major mediator of angiogenesis, a critically important process in vertebrate growth and development as well as pregnancy. Here we report for the first time the expression of a rare and unusually potent splice variant, VEGF 111 , in vivo in mammals. This variant has previously only been found in mammals in cultured human cells exposed to genotoxic agents. Our discovery of VEGF 111 in the uterus of both a eutherian (rat) and a marsupial (fat-tailed dunnart) suggests that the splice variant may be common to all mammals. As VEGF 111 is also expressed in the uterus of at least one lineage of lizards, the expression of this splice variant may be a widespread amniote phenomenon. We measured expression of VEGF 111 and two major VEGF-A splice variants in the uterus of pregnant rats, showing that the three variants show different expression patterns across pregnancy. Our results suggest that viviparous mammals possess a precisely regulated milieu of VEGF isoforms producing the angiogenesis required for successful pregnancy. The discovery of VEGF 111 in rat uterus paves the way for the development of in vivo models of VEGF 111 activity in a highly tractable laboratory animal, and is particularly significant in the context of early pregnancy loss and cancer research.

During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web.

During early pregnancy, the uterine luminal epithelial cells (UECs) and endometrial stromal cells (ESCs) undergo morphological changes to enable blastocyst implantation. The present study investigates, for the first time, the cytoskeletal-associated proteins and α-actinin superfamily members, α-parvin and β-parvin, during early pregnancy in the rat uterus. These two PARVA proteins are involved in cell adhesion, morphological changes and regulation of other cytoskeletal proteins, through binding with proteins such as actin and integrin-linked kinase. α-parvin is present in UECs at fertilisation and significantly decreases by the time of implantation. β-parvin acts in opposition; significantly increasing in both UECs and ESCs at the time of implantation, suggesting a role in the process of decidualisation. Additionally, the presence of a serine-8 residue-phosphorylated α-parvin, which is associated with cell morphology changes, was found in the nuclear region of both UECs and ESCs during implantation and decidualisation. We also show that the presence of both β-parvin and phosphorylated α-parvin in ESCs is dependent on decidualisation occurring. This study demonstrates that the changing balance and localisation of the two PARVA proteins are dependent on the time of uterine receptivity, suggesting a co-dependent role in the cytoskeletal re-organisation crucial to the changing conditions necessary for implantation and decidualisation.

The uterine surface undergoes significant remodeling, termed the "plasma membrane transformation," during pregnancy to allow for implantation of the blastocyst and formation of the placenta in viviparous amniote vertebrates. Unlike other species within the superorder Euarchontoglires, which have a hemochorial (highly invasive) placenta, kangaroo rats (Dipodomys spp.) exhibit a less invasive endotheliochorial placenta. We characterized the changes that occur to membrane molecules and the cellular ultrastructure of the uterine epithelium during early pregnancy in Merriam's kangaroo rat, Dipodomys merriami using electron microscopy and immunofluorescence microscopy. Epithelial cadherin (E-cadherin) is an adhesion protein that forms the adherens junction and is localized to the lateral plasma membrane of uterine epithelium during the nonreproductive state but localizes nonspecifically in the uterine epithelium immediately preceding implantation. Desmosomes are a type of cadherin that form junctional complexes along the lateral plasma membrane of epithelium. Dsg-2, a marker for desmosomes, is localized along the lateral plasma membrane in non-pregnant animals but redistributes to the apical region of the lateral plasma membrane during early pregnancy. The shift in desmosome and cadherin distribution before implantation suggests that there is a reduction in lateral adhesion between epithelial cells to allow for invasion by the blastocyst. Surprisingly, although Kangaroo rats form a less invasive placenta, these same changes occur during pregnancy in species with highly invasive placentation, such as the laboratory rat and human. These commonalities suggest that it is not through the retention of lateral adhesion that the blastocyst is prevented from further invasion in this rodent species. Anat Rec, 301:1928-1935, 2018. © 2018 Wiley Periodicals, Inc.

Mammals exhibit similar changes in uterine epithelial morphology during early pregnancy despite having a diverse range of placental types. The uterine epithelium undergoes rapid morphological and molecular change ("plasma membrane transformation") during the early stages of pregnancy to allow attachment of the blastocyst. The domestic cat, Felis catus is in the order Carnivora; all species within the Carnivora studied so far develop an endotheliochorial placenta during pregnancy. The endotheliochorial placental type is a common form of placental invasion in mammals. The molecular changes that allow remodeling of the uterine epithelium in preparation for implantation are unknown in most mammals but would provide us with an understanding of what molecules underpin successful implantation and pregnancy among Carnivora. We used immunofluorescence microscopy to localize the key adherens junction proteins desmoglein-2 and E-cadherin in the lateral plasma membrane of the uterine epithelium of F. catus during pregnancy. We show that redistribution of desmoglein-2 and E-cadherin likely facilitates reduction of cell-to-cell adhesion allowing for implantation of the blastocyst and formation of the placenta. The ultrastructural and molecular changes to the uterine epithelium during early pregnancy in F. catus are similar to that in species with other levels of placental invasiveness, suggesting that key molecules such as desmoglein-2 and E-cadherin are crucial to successful pregnancy across all mammals. Anat Rec, 301:1497-1505, 2018. © 2018 Wiley Periodicals, Inc.

The evolution of viviparity requires the development of mechanisms that facilitate transport of respiratory gases between mother and developing embryo. Of particular importance is maternal excretion of embryonic carbon dioxide (CO2), which increases as the embryo grows in size during development. The carbonic anhydrases are a family of enzymes that convert CO2 to bicarbonate for transport throughout the cardiovascular system and which may also be important for CO2 transport from embryo to mother. We used immunohistochemistry to localize carbonic anhydrase II in the placental tissues of a viviparous and highly placentotrophic lizard, Pseudemoia entrecasteauxii. Carbonic anhydrase II is localized in the uterine component of the paraplacentome, presumably to facilitate transport of embryonic CO2 to the mother. Carbonic anhydrase II is also localized in both the uterine and embryonic components of the placentome, a region heavily involved in placental nutrient transport rather than respiratory gas exchange. In contrast, carbonic anhydrase II is not present in the uterine or embryonic components of the omphaloplacenta, another region responsible for nutrient transport. While carbonic anhydrase II in the paraplacentomal uterus is likely to be responsible for embryo-maternal CO2 transport, the distribution of carbonic anhydrase II throughout the placentome indicates a different function. Instead of transporting embryonic CO2, placentomal carbonic anhydrase II appears to be responsible for transporting CO2 produced by energetically expensive nutrient transport mechanisms in both the uterus and the embryo, which implies that the mechanisms of nutrient transport in the omphaloplacenta may not be as energetically expensive. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 636–646, 2015. © 2015 Wiley Periodicals, Inc.

Angiogenesis (blood vessel growth), a key process of mammalian pregnancy, facilitates gas exchange and nutrient transport between the mother and the embryo and is regulated by a suite of growth factors. Vascular endothelial growth factor (VEGF) is crucial to this process in pregnant mammals and potentially pregnant squamates (lizards and snakes), as we investigate here. VEGF111, an unusual and potent angiogenic splice variant of VEGF, increases its expression during pregnancy in the uterus of a viviparous lizard, in parallel with similar increases in uterine angiogenesis during gestation. However, we also find that VEGF111 is expressed in oviparous skinks, and is not ubiquitous among viviparous skinks. Thus, different mechanisms of uterine angiogenesis during pregnancy may evolve concurrent with viviparity in different lizard lineages. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 152–158, 2015. © 2015 Wiley Periodicals, Inc.

Introduction Transrectal ultrasound guided prostate biopsy may be performed by operators with various levels of training. Little is known about the impact of training level on biopsy results. We evaluated the effect of training level on the accuracy of transrectal ultrasound guided prostate biopsy findings. Methods We retrospectively reviewed 500 consecutive patients who underwent transrectal ultrasound guided prostate biopsy and subsequent radical prostatectomy. Transrectal ultrasound operators were stratified based on level of training as junior, senior, chief, fellow or staff. Linear regression was performed to analyze the effect of training level on volume estimates. A weighted Kappa statistic evaluated agreement between biopsy and pathological Gleason scores while an adjusted cumulative logistic regression model analyzed the effects of training level. Results A total of 482 patients were included in the final analysis. Transrectal ultrasound guided biopsy was performed by staff in 78 (16%) patients, by fellows in 18 (4%), chief residents in 48 (10%), senior residents in 126 (26%) and junior residents in 212 (44%). There was no significant difference between transrectal ultrasound and radical prostatectomy specimen volume estimates among the training levels. Level of training was not significantly associated with pathological features, including Gleason score, primary Gleason grade, highest single Gleason grade and estimated tumor volume. Study limitations include the retrospective design and the variability among members of the same group. Conclusions Agreement between biopsy and pathological Gleason scores is high for all levels of training. Training level has no impact on prostate volume estimations or the prediction of pathological features.

The plasma membrane of uterine epithelial cells undergoes a number of changes during early pregnancy. The changes in the basolateral membrane at the time of implantation in particular change from being smooth to highly tortuous in morphology, along with a dramatic increase in the number of morphological caveolae at this time. The major protein of caveolar membranes is caveolin, and previous studies have shown that RNA pol I transcription factor (PTRF) and serum deprivation protein response (SDPR) are the two members of the cavin protein family. These proteins are known to be involved in caveolae biogenesis, where they directly bind to cholesterol and lipids and have been reported to promote membrane curvature. As there is an increase in membrane tortuosity and caveolae at the time of implantation, this study investigated PTRF and SDPR to explore the possible roles that they play in the morphology of the uterine epithelium during early pregnancy. PTRF protein abundance did not change in uterine epithelial cells during early pregnancy or in response to ovarian hormones. At the time of implantation in uterine epithelial cells, PTRF co-immunoprecipitated with caveolin 1, thereby demonstrating an association with caveolin-1 at the basal plasma membrane in caveolae. SDPR protein was observed to be present only at the time of fertilisation, and also under the influence of oestrogen alone, where a cytoplasmic localisation in uterine epithelial cells was observed. The localisation and expression PTRF and SDPR in uterine epithelial cells during early pregnancy suggest that they have roles in the maintenance of lipids and cholesterol in the plasma membrane. PTRF and lack of SDPR may contribute not only to the morphology of the basal plasma membrane as observed at the time of implantation, but also to the maintenance of epithelial polarity during early pregnancy.

We report salvage lymph node dissections for prostate cancer nodal recurrence detected by (11)C-choline positron emission tomography/computerized tomography in the setting of increasing prostate specific antigen after radical prostatectomy.Retrospective chart review was performed for all patients who underwent salvage lymph node dissection for prostate cancer nodal recurrence. Only patients previously treated with radical prostatectomy were included in the study and those with evidence of local recurrence were excluded from analysis. Primary end points included biochemical recurrence, systemic progression and cancer specific mortality.From 2009 to 2013, 52 men underwent salvage lymph node dissection. Before salvage lymph node dissection 78.8% (41 of 52) had some form of therapy after radical prostatectomy. Median age at salvage lymph node dissection was 60 years and median prostate specific antigen was 2.2 ng/ml (IQR 1.4-3.7). The median number of lymph nodes dissected was 21.5 (IQR 16-30) and the median number of positive nodes was 3.5 (IQR 1.2-6.5). Since salvage lymph node dissection 46.2% of the men (24 of 52) have had no further treatment, 34.6% (18 of 52) are on hormonal therapy and 19.2% (10 of 52) have received multiple different treatments. At the last followup at a median of 20 months (IQR 8-33), 57.7% (30 of 52) had prostate specific antigen remain less than 0.2 ng/ml, 75% (39 of 52) remained free of systemic progression and 96.2% of the men (50 of 52) were alive. Two patients died of prostate cancer. Three-year biochemical recurrence-free, systemic progression-free and cancer specific survival was 45.5%, 46.9% and 92.5%, respectively.This represents the largest U.S. series of salvage lymph node dissection in the setting of lymph node metastatic prostate cancer after radical prostatectomy. Although followup was short and the study lacked a randomized control group, salvage lymph node dissection may represent a valid treatment option.

The epithelium of the uterine lumen is the first point of contact with the blastocyst before implantation. To facilitate pregnancy, these uterine epithelial cells (UECs) undergo morphological changes specific to the receptive uterus. These changes include basal, lateral and apical alterations in the plasma membrane of UECs. This study looked at the cytoskeletal and focal adhesion-associated proteins, lasp-1 and palladin, in the uterus during early pregnancy in the rat. Two palladin isoforms, 140 kDa and 90 kDa, were analysed, with the migration-associated 140-kDa isoform increasing significantly at the time of implantation when compared with the time of fertilisation. Lasp-1 was similarly increased at this time, whilst also being located predominantly apically and laterally in the UECs, suggesting a role in the initial contact between the UECs and the blastocyst. This is the first study to investigate palladin and lasp-1 in the uterine luminal epithelium and suggests an importance for these cytoskeletal proteins in the morphological changes the UECs undergo for pregnancy to occur.

During early pregnancy, uterine epithelial cells (UECs) become less adherent to the underlying basal lamina and are subsequently removed so the blastocyst can invade the underlying stroma. This process involves the removal of focal adhesions from the basal plasma membrane of UECs. These focal adhesions are thought to be internalized by caveolae, which significantly increase in abundance at the time of blastocyst implantation. A recent in vitro study indicated that prominin-2 prevents the formation of caveolae by sequestering membrane cholesterol. The present study examines whether prominin-2 affects the formation of caveolae and loss of focal adhesions in UECs during normal and ovarian hyperstimulation (OH) pregnancy in the rat. At the time of fertilization during normal pregnancy, prominin-2 is distributed throughout the basolateral plasma membrane. However, at the time of implantation and coincident with an increase in caveolae, prominin-2 is lost from the basal plasma membrane. In contrast, prominin-2 remains in the basolateral plasma membrane throughout OH pregnancy. Transmission electron microscopy showed that this membrane contained few caveolae throughout OH pregnancy. Our results indicate that prominin-2 prevents the formation of caveolae. We suggest the retention of prominin-2 in the basal plasma membrane during OH pregnancy prevents the formation of caveolae and is responsible for the retention of focal adhesions in this membrane, thereby contributing to the reduced implantation rate observed after such treatments.

The evolution of viviparity requires eggshell thinning to bring together the maternal uterus and extraembryonic membranes to form placentae for physiological exchanges. Eggshell thinning likely involves reduced activity of the uterine glands that secrete it. We tested these hypotheses by comparing the uterine and eggshell structure and histochemistry among oviparous and viviparous water snakes (Helicops) using phylogenetic methods. Eggshell thinning occurred convergently in all three origins of viviparity in Helicops and was accomplished by the loss of the mineral layer and thinning of the shell membrane. Uterine glands secrete the shell membrane in both oviparous and viviparous Helicops. These glands increase during vitellogenesis regardless of the reproductive mode, but they always reach smaller sizes in viviparous forms. As there is no phylogenetic signal in eggshell thickness and gland dimensions, we conclude that interspecific differences are related to reproductive mode and not phylogeny. Therefore, our results support the hypothesis that eggshell thinning is associated with the evolution of viviparity and that such thinning result from a reduction in gland size in viviparous taxa. Interestingly, the shell membrane thickness of viviparous females of the reproductively bimodal Helicops angulatus is intermediate between their oviparous and viviparous congeners. Thus, although eggshell thinning is required by the evolution of viviparity, a nearly complete loss of this structure is not. However, uterine gland dimensions are similar across viviparous Helicops. Fewer glands or their functional repurposing may explain the thinner shell membrane in viviparous species of Helicops in comparison to viviparous females of the bimodal H. angulatus.

In mammalian pregnancy, the uterus is remodeled to become receptive to embryonic implantation. Since non-invasive placentation in marsupials is likely derived from invasive placentation, and is underpinned by intra-uterine conflict between mother and embryo, species with non-invasive placentation may employ a variety of molecular mechanisms to maintain an intact uterine epithelium and to prevent embryonic invasion. Identifying such modifications to the uterine epithelium of marsupial species with non-invasive placentation is key to understanding how conflict is mediated during pregnancy in different mammalian groups. Desmoglein-2, involved in maintaining lateral cell-cell adhesion of the uterine epithelium, is redistributed before implantation to facilitate embryo invasion in mammals with invasive placentation. We identified localization patterns of this cell adhesion molecule throughout pregnancy in two marsupial species with non-invasive placentation, the tammar wallaby (Macropus eugenii; Macropodidae), and the brushtail possum (Trichosurus vulpecula; Phalangeridae). Interestingly, Desmoglein-2 redistribution also occurs in both M. eugenii and T. vulpecula, suggesting that cell adhesion, and thus integrity of the uterine epithelium, is reduced during implantation regardless of placental type, and may be an important component of uterine remodeling. Desmoglein-2 also localizes to the mesenchymal stromal cells of M. eugenii and to epithelial cell nuclei in T. vulpecula, suggesting its involvement in cellular processes that are independent of adhesion and may compensate for reduced lateral adhesion in the uterine epithelium. We conclude that non-invasive placentation in marsupials involves diverse and complementary strategies to maintain an intact epithelial barrier.

The uterine luminal epithelium is the first site of contact between fetal and maternal tissues during therian pregnancy and must undergo specialised changes for implantation of the blastocyst to be successful. These changes, collectively termed the plasma membrane transformation (PMT), allow the blastocyst to attach to the uterine epithelium preceding the formation of a placenta. There are similarities in the morphological and molecular changes occurring in live-bearing eutherian species during the PMT studied so far. Similar cellular remodelling occurs in a marsupial species, the fat-tailed dunnart (Sminthopsis crassicaudata), despite the divergence of marsupials from eutherian mammals over 130 mya, which resulted in the evolution of distinct reproductive strategies. Adhesion molecules along the lateral plasma membrane of uterine epithelium provide a barrier to invasion by the embryo. We thus characterised the presence and change in distribution of epithelial cadherin (E-cadherin) in uterine epithelium from non-pregnant fat-tailed dunnarts and compared it to dunnarts in early-, mid- and late-stage pregnancy. E-cadherin staining is localised to the lateral plasma membrane in uterine epithelium from non-pregnant and early-stage pregnant dunnarts. The E-cadherin staining is cytoplasmic in epithelium from uteri of mid- and late-stage pregnant dunnarts. This loss of localised staining suggests that the adherens junction dissociates from the lateral plasma membrane, allowing for invasion between the epithelial cells by the blastocyst. As the changes during pregnancy to cadherin were similar in the laboratory rat with highly invasive (haemochorial) placentation, a live-bearing lizard species with non-invasive (epitheliochorial) placentation and a marsupial, the fat-tailed dunnart, which has invasive (endotheliochorial) placentation, we suggest that the molecular mechanisms allowing for successful pregnancy are conserved among mammals during the early stages of pregnancy regardless of placental invasiveness.

Pregnancy in mammals requires remodeling of the uterus to become receptive to the implanting embryo. Remarkably similar morphological changes to the uterine epithelium occur in both eutherian and marsupial mammals, irrespective of placental type. Nevertheless, molecular differences in uterine remodeling indicate that the marsupial uterus employs maternal defences, including molecular reinforcement of the uterine epithelium, to regulate embryonic invasion. Non-invasive (epitheliochorial) embryonic attachment in marsupials likely evolved secondarily from invasive attachment, so uterine defences in these species may prevent embryonic invasion. We tested this hypothesis by identifying localization patterns of Talin, a key basal anchoring molecule, in the uterine epithelium during pregnancy in the tammar wallaby (Macropus eugenii; Macropodidae) and the brush tail possum (Trichosurus vulpecula; Phalangeridae). Embryonic attachment is non-invasive in both species, yet Talin undergoes a clear distributional change during pregnancy in M. eugenii, including recruitment to the base of the uterine epithelium just before attachment, that closely resembles that of invasive implantation in the marsupial species Sminthopsis crassicaudata. Basal localization occurs throughout pregnancy in T. vulpecula, although, as for M. eugenii, this pattern is most specific prior to attachment. Such molecular reinforcement of the uterine epithelium for non-invasive embryonic attachment in marsupials supports the hypothesis that less-invasive and non-invasive embryonic attachment in marsupials may have evolved via accrual of maternal defences. Recruitment of basal molecules, including Talin, to the uterine epithelium may have played a key role in this transition.

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non-invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non-invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.

Following mating, leukocytes are recruited to the uterine epithelium where they phagocytose spermatozoa and mediate maternal immune tolerance as well as a mild inflammatory response. In this ultrastructural study we utilised array tomography, a high-resolution volume scanning electron microscopy approach to 3D reconstruct the cellular relationships formed by leukocytes recruited to the luminal uterine epithelium 12 h post-mating in the rat. We report that following mating, neutrophils and macrophages are internalised by the luminal uterine epithelium, with multiple leukocytes internalised via contortion through a small tunnel in the apical membrane into a large membrane-bound vacuole within the cytoplasm of luminal uterine epithelial cells (UECs). Once internalised within the UECs, recruited leukocytes appear to phagocytose material within the membrane-bound vacuole and most ultimately undergo a specialised cell death, including vacuolisation and loss of membrane integrity. As these observations involve ultrastructurally normal leukocytic cells internalised within non-phagocytic epithelial cells, these observations are consistent with the formation of cell-in-cell structures via entosis, rather than phagocytic engulfment by UECs. Although cell-in-cell structures have been reported in normal and pathological conditions elsewhere, the data collected herein represents the first evidence of the formation of cell-in-cell structures within the uterine epithelium as a novel component of the maternal inflammatory response to mating.

The non-receptive uterine luminal epithelium forms a polarised epithelial barrier, protective against potential pathogenic assault from the external environment and invasion by the blastocyst. However, during the window of implantation, the uterine luminal epithelial cells (UECs) transition to a receptive state by dismantling many of their intercellular and cell-matrix adhesions in preparation for epithelial detachment and subsequent blastocyst implantation. The present study investigated the presence and regulation of the intercellular adhesion protein, Epithelial Cell Adhesion Molecule (EpCAM) during early pregnancy in the rat to understand its role in the transition to receptivity. Immunofluorescence and western blotting analysis were used to study EpCAM expression in normal pregnancy, hormone replacement studies and pseudopregnancy. EpCAM was abundantly expressed and localised to the uterine luminal and glandular epithelium during the non-receptive state but decreased to lower but still observable levels around the time of implantation. This decrease was not dependent on ovarian hormones or the blastocyst. Further, EpCAM colocalised with but did not associate with its frequent binding partner, Tumour necrosis factor α (TNFα)-converting enzyme, also known as A Disintegrin And Metalloprotease 17 (TACE/ADAM17), at the time of fertilisation. These results suggest that, prior to implantation, EpCAM mediates intercellular adhesion in the uterine epithelium, but that, during implantation when UECs lose the majority of their intercellular and cell-matrix adhesions, EpCAM levels are decreased but still present for the maintenance of mucosal integrity.

Attachment of the blastocyst and formation of the placenta during pregnancy is dependent on structural and cellular changes occurring in the uterine epithelium and in particular to the plasma membrane of these uterine cells. Desmosome expression decreases during pregnancy in eutherians and some squamates, presumably allowing for remodeling of the uterine epithelium and invasion of the trophoblast during implantation. Marsupials are a distinct mammalian amniote lineage of viviparity, with a short implantation or attachment period and varying levels of invasive placentation. To test the generality of changes to the uterine epithelium during pregnancy across mammals, we characterized the distribution of desmosomes in the uterine epithelial cells of a marsupial, Sminthopsis crassicaudata, using electron microscopy and immunohistochemistry. The absolute number of desmosomes along the lateral plasma membrane decreases during pregnancy and desmosomes are redistributed towards the apical region of the lateral plasma membrane as pregnancy proceeds, similar to what occurs during pregnancy in eutherian mammals. Despite the lower level of maternal investment in pregnancy and the noninvasive structure of fetal membranes in marsupials there are similarities in number and redistribution of desmosomes along the plasma membrane and changes to the morphology of the uterine epithelial cells suggesting that similar plasma membrane changes occur across all lineages of amniote vertebrates.

At the time of implantation, uterine luminal epithelial cells undergo a dramatic change in all plasma membrane domains. Changes in the basolateral plasma membrane at the time of implantation include progression from smooth to highly tortuous, as well as a loss of integrin-based focal adhesions. Another aspect of the basolateral plasma membrane that has not been studied in uterine epithelial cells are caveolae, which are omega-shaped invaginations of the plasma membrane known to be involved in endocytosis and contribute to membrane curvature. The current study investigated caveolin, a major protein of caveolae, to explore the possible roles that they play in the remodelling of the basolateral plasma membrane of uterine epithelial cells during early pregnancy in the rat. Morphological caveolae were found at the time of implantation and were significantly increased compared to day 1 of pregnancy. Caveolins 1 and 2 were found to shift to the basolateral plasma membrane of uterine epithelial cells at the time of implantation as well as when treated with progesterone alone, and in combination with oestrogen. A statistically significant increase in the amount of caveolin-1 and a decrease in caveolin-2 protein in uterine epithelial cells was observed at the time of implantation. Caveolin-1 also co-immunoprecipitated with integrin β1 on day 1 of pregnancy, which is a protein that has been reported to be found in integrin-based focal adhesions at the basolateral membrane on day 1 of pregnancy. The localisation and expression of caveolin-1 at the time of implantation is consistent with the presence and increase of morphological caveolae seen at this time. The localisation and expression of caveolins 1 and 2 in luminal uterine epithelium at the time of implantation suggest a role in trafficking proteins and the maintenance of a polarised epithelium.

Formation of a placenta requires intimate contact between the embryonic and maternal uterine epithelia in early pregnancy. Contact is accompanied by a characteristic suite of changes to the plasma membranes of uterine epithelial cells, termed the plasma membrane transformation. The plasma membrane transformation occurs in eutherian mammals and in viviparous (live-bearing) squamate reptiles, and may be fundamental to the evolution of viviparity in amniotes. Marsupials provide an excellent opportunity to test the generality of this phenomenon. Here, we present the first detailed study of the plasma membrane transformation in a marsupial. We combine electron microscopy and immunohistochemistry to describe morphological and molecular features of uterine epithelial cells during pregnancy in the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Cell morphology changes dramatically in S. crassicaudata during pregnancy. Apical microvilli are replaced by irregular blunt projections, then by spiky projections postimplantation. Cell surfaces flatten and ciliated cells are lost. Junctional complexes between adjacent cells increase in depth, then decrease just before implantation, which is consistent with junctional protein localization in this region of the cell membrane. The uterine cellular changes in S. crassicaudata are consistent with a plasma membrane transformation, and support the idea that this phenomenon is fundamental to the evolution of viviparity in amniote vertebrates.

The present study investigated the role of calpain 2 in rat uterine luminal epithelial cells during early pregnancy. Calpain 2 is an intracellular calcium-dependent proteolytic enzyme which cleaves numerous focal adhesion proteins. Calpain 2 was concentrated along the basal cell surface of uterine luminal epithelial cells at the predicted site of focal adhesions on day 1 of pregnancy and remained unchanged at the time of implantation as observed by immunofluorescence microscopy. However, Western blotting analysis showed a marked increase in the active form and a significant decrease in the latent form of calpain 2 at the time of implantation. The increase in calpain 2 activity coincides with the disassembly of focal adhesion proteins, talin, paxillin, integrin β1 and β3 from the site of focal adhesions. Intraperitoneal injection of calpain inhibitor, calpain inhibitor l (ALLN), significantly reduced the number of implantation sites, implying that calpain 2 plays an important role in implantation. The present study suggests a role for calpain 2 in the disassembly of focal adhesions, which has been previously shown to play a key role in uterine receptivity for implantation.

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.

Vascular endothelial growth factor A is a major mediator of angiogenesis, a critically important process in vertebrate growth and development as well as pregnancy. Here we report for the first time the expression of a rare and unusually potent splice variant, VEGF

The non-receptive uterine luminal epithelium forms an intact polarised epithelial barrier that is refractory to blastocyst invasion. During implantation, organised dismantling of this barrier leads to a receptive state promoting blastocyst attachment. Claudins are tight junction proteins that increase in the uterine epithelium at the time of implantation. Claudin 7 is a member of this family but demonstrates a basolateral localisation pattern that is distinct from other claudins. The present study investigated the localisation, abundance and hormonal regulation of claudin 7 to elucidate a role for the protein during implantation. The results showed that claudin 7 demonstrates a distinct basal and lateral localisation in the uterine luminal and glandular epithelium throughout early pregnancy. On day 1, claudin 7 is abundantly present in response to ovarian estrogen. At the time of implantation, claudin 7 decreases in abundance. This decrease is not dependent on blastocyst presence, as shown by results in pseudopregnant animals. We propose that claudin 7 mediates intercellular adhesions in the uterine epithelium and also may be responsible for stabilising adhesion proteins at the basolateral cell surface. Thus, claudin 7 may function under the maintenance of the uterine luminal epithelial barrier, in the non-receptive state preventing implantation from occurring.

Integrins are expressed in a highly regulated manner at the maternal-fetal interface during implantation. However, the significance of extracellular matrix (ECM) ligands during the integrin-mediated embryo attachment to the endometrium is not fully understood. Thus, the distribution of fibronectin in the rat uterus and blastocyst was studied at the time of implantation. Fibronectin was absent in the uterine luminal epithelial cells but was intensely expressed in the trophoblast cells and the inner cell mass suggesting that fibronectin secreted from the blastocyst may be a possible bridging ligand for the integrins expressed at the maternal-fetal interface. An Arg-Gly-Asp (RGD) peptide was used to block the RGD recognition sites on integrins, and the effect on rat blastocyst attachment to Ishikawa cells was examined. There was a significant reduction in blastocyst attachment when either the blastocysts or the Ishikawa cells were pre-incubated with the RGD-blocking peptide. Thus, successful attachment of the embryo to the endometrium requires the interaction of integrins on both the endometrium and the blastocyst with the RGD sequence of ECM ligands, such as fibronectin. Pre-treatment of both blastocysts and Ishikawa cells with the RGD peptide also inhibited blastocyst attachment, but not completely, suggesting that ECM bridging ligands that do not contain the RGD sequence are also involved in embryo attachment. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.

Frequent evolutionary changes in reproductive mode have produced a wide range of placental structures in viviparous squamate reptiles. Closely related species with different placental structures and resolved phylogenetic relationships are particularly useful for reconstructing how placentae might have transformed during the evolutionary process. We used light microscopy to study placental morphology in mid- to late stage embryos of four closely related species of Eulamprus, a genus of viviparous scincid lizards that we had reason to suspect may display significant interspecific variation in placental morphology. Embryos from all four species possess a chorioallantoic placenta, an omphaloplacenta and an interomphalopleuric membrane, characteristics present in other viviparous skinks. However, unlike other viviparous skinks but characteristic of oviparous skinks, the allantois expands to surround the yolk sac in each species, supplanting the omphalopleure with a larger area of chorioallantois until a chorioallantoic placenta surrounds the entire egg in one specimen that is only a few days from birth. All four Eulamprus species share the same extraembryonic membrane morphology, but the cellular morphology of the uterine epithelium in the chorioallantoic placenta and omphaloplacenta varies between species. We determined that the interomphalopleuric membrane is a shared derived character of the Eulamprus quoyii species group. New phylogenetic information indicates that variation in the chorioallantoic placenta is a result of two independent transitions, but that variation in the omphaloplacenta can be explained using a single change within the species studied. Our results indicate that E. quoyii group skinks are a valuable model for investigating the evolution of viviparity, as extraembryonic membrane development in these species shows features characteristic of both oviparous and viviparous skinks.

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established. J. Exp. Zool. (Mol. Dev. Evol.) 318:148–158, 2012. © 2012 Wiley Periodicals, Inc.