Search

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.

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.

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.

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.

© 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.

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.

The 2018 edition of the Society for Reproductive Biology’s (SRB) Annual Meeting was a celebration of 50 years of Australian research into reproductive biology. The past 50 years has seen many important contributions to this field, and these advances have led to changes in practice and policy, improvements in the efficiency of animal reproduction and improved health outcomes. This conference review delivers a dedicated summary of the symposia, discussing emerging concepts, raising new questions and proposing directions forward. Notably, the symposia discussed in this review emphasised the impact that reproductive research can have on quality of life and the health trajectories of individuals. The breadth of the research discussed encompasses the central regulation of fertility and cyclicity, life course health and how the environment of gametes and embryos can affect subsequent generations, significant advances in our understanding of placental biology and pregnancy disorders and the implications of assisted reproductive technologies on population health. The importance of a reliable food supply and protection of endangered species is also discussed. The research covered at SRB’s 2018 meeting not only recognised the important contributions of its members over the past 50 years, but also highlighted key findings and avenues for innovation moving forward that will enable the SRB to continue making significant contributions for the next 50 years.

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.

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.

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.

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.

During early pregnancy the endometrium undergoes a major transformation in order for it to become receptive to blastocyst implantation. The actin cytoskeleton and plasma membrane of luminal uterine epithelial cells (UECs) and the underlying stromal cells undergo dramatic remodelling to facilitate these changes. Filamin A (FLNA), a protein that crosslinks actin filaments and also mediates the anchorage of membrane proteins to the actin cytoskeleton, was investigated in the rat uterus at fertilisation (Day 1) and implantation (Day 6) to determine the role of FLNA in actin cytoskeletal remodelling of UECs and decidua during early pregnancy. Localisation of FLNA in UECs at the time of fertilisation was cytoplasmic, whilst at implantation it was distributed apically; its localisation is under the influence of progesterone. FLNA was also concentrated to the first two to three stromal cell layers at the time of fertilisation and shifted to the primary decidualisation zone at the time of implantation. This shift in localisation was found to be dependent on the decidualisation reaction. Protein abundance of the FLNA 280-kDa monomer and calpain-cleaved fragment (240 kDa) did not change during early pregnancy in UECs. Since major actin cytoskeletal remodelling occurs during early pregnancy in UECs and in decidual cells, the changing localisation of FLNA suggests that it may be an important regulator of cytoskeletal remodelling of these cells to allow uterine receptivity and decidualisation necessary for implantation in the rat.

In preparation for uterine receptivity, the uterine epithelial cells (UECs) exhibit a loss of microvilli and glycocalyx and a restructuring of the actin cytoskeleton. The prominin-1 protein contains large, heavily glycosylated extracellular loops and is usually restricted to apical plasma membrane (APM) protrusions. The present study examined rat UECs during early pregnancy using immunofluorescence, western blotting and deglycosylation analyses. Ovariectomised rats were injected with oestrogen and progesterone to examine how these hormones affect prominin-1. At the time of fertilisation, prominin-1 was located diffusely in the apical domain of UECs and 147- and 120-kDa glycoforms of prominin-1 were identified, along with the 97-kDa core protein. At the time of implantation, prominin-1 concentrates towards the APM and densitometry revealed that the 120-kDa glycoform decreased (P

1. Sabcomeline (SB-202026, 0.03 mg kg(-1), p.o.), a potent and functionally selective M1 receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg(-1), p.o.). 2. Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg(-1), p.o.). 3. Sabcomeline (0.03 and 0.1 mg kg(-1), p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset. 4. Sabcomeline (0.03 mg kg(-1), p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking. 5. This is the first study to demonstrate cognitive enhancement by a functionally selective M1 receptor partial agonist in a normal (i.e. non-cognitively impaired) non-human primate and this effect was seen at a dose which did not cause side effects. 6. Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer's disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.