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Brook Trout (Salvelinus fontinalis) have been widely introduced throughout the world and are often considered as direct competitors with native salmonid species. Metabolic rate is one metric we can examine to improve our understanding of how well fish perform in different habitats, including across temperature gradients, as metabolism can be directly influenced by environmental temperatures in ectotherms. We estimated the standard metabolic rate, maximum metabolic rate, and aerobic scope of lab-reared juvenile Brook Trout (~1 year) using intermittent-flow respirometry across a range of temperatures (5–23 °C) likely experienced in the wild. We included a diurnal temperature cycle of ±1.5 °C for each treatment temperature to simulate temporal variation observed in natural waterbodies. Standard metabolic rate and maximum metabolic rate both increased with acclimation temperature before appearing to plateau around 20 °C, while mass specific aerobic scope was found to increase from a mean of 287.25 ± 13.03 mg O2·kg-1·h-1 at 5 °C to 384.85 ± 13.31 mg O2·kg-1·h-1 at 15 °C before dropping at higher temperatures. Although a slight peak was found at 15 °C, the generally flat thermal performance curve for aerobic scope suggests Brook Trout are capable of adjusting to a relatively wide range of thermal regimes, appearing to be eurythermal, or a thermal generalist, at least for salmonids. The ability of this population to maintain similar physiological performance across a wide range of temperatures may help explain why Brook Trout succeed in a variety of different thermal habitats.

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at −80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlaterup to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA’s Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

The way in which maternal blood is associated with trophoblast prior to the formation of the different types of hemochorial placenta may be conveniently grouped into four main patterns: a transitory endotheliochorial condition; maternal blood released into a mass of trophoblast; maternal blood confined to lacunae; and fetal villi entering preexisting maternal blood sinuses. Although it might be considered logical that developing placentas would pass through an endotheliochorial stage to become hemochorial, this developmental pattern is seen only as a transient stage in several species of bats and sciuromorph rodents. More commonly a mass of trophoblast at the junction with the endometrium serves as a meshwork through which maternal blood passes, with subsequent organization of a labyrinth when the fetal vascular component is organized. The initial trophoblast meshwork may be cellular or syncytial, often leading to a similar relationship in the spongy zone and labyrinth. Old World monkeys, apes and humans have a lacunar stage prior to establishing a villous hemochorial condition. New World monkeys lack a true lacunar stage, retaining portions of maternal vessels for some time and initially forming a trabecular arrangement similar to though differently arrived at than that in the tarsier. In armadillos, preexisting maternal venous sinuses are converted into an intervillous blood space by intruding fetal villi. Variations from the major patterns of development also occur. The way in which the definitive placental form is achieved developmentally should be considered when using placental structure to extrapolate evolution of placentation.

Udgivelsesdato: 2009-Jul The placentation of the Hottentot golden mole (Amblysomus hottentotus) has been examined using light and electron microscopy and lectin histochemistry of nine specimens at both mid and late gestation. The placentae were lobulated towards the allantoic surface and the lobules contained roughly parallel arrays of labyrinthine structures converging on a central spongy zone. At mid gestation, the arrays were composed of an inner cellular and outer syncytial trophoblast layer, the inner layer enclosing scant connective tissue and fetal capillaries. Maternal blood spaces coursed through the outer trophoblast and were lined by trophoblastic microvilli; the blood spaces were narrow in mid gestation but enlarged near term, while the inner trophoblast layer became thinner and seemed to be syncytial. These features were confirmed by transmission electron microscopy. The microvillous surfaces and dispersed cytoplasmic particles were heavily glycosylated, as shown by lectin histochemistry, and exhibited changes with maturation, particularly a loss in N-acetyl glucosamine oligomers bound by Phytolacca americana lectin on the microvilli lining the maternal blood spaces and outer trophoblast particles. A substantial yolk sac was present both in mid and late gestation stages. It was clearly unattached to the uterus in the later stages. These morphological features are discussed in relation to the phylogenetic position of Amblysomus with respect to other members of Afrosoricida and Afrotheria.

BACKGROUND: Transmission electron microscopy (TEM) was first applied to study placental ultrastructure in the 1950's. We review those early studies and mention the scientists that employed or encouraged the use of TEM.FINDINGS: Among the pioneers Edward W. Dempsey was a key figure who attracted many other scientists to Washington University in St. Louis. Work on human placental ultrastructure was initiated at Cambridge and Kyoto whilst domestic animals were initially studied by Björkman in Stockholm and electron micrographs of bat placenta were published by Wimsatt of Cornell University.CONCLUSIONS: Prior to the introduction of better fixation techniques, TEM images were of modest technical quality. Nevertheless they gave important insights into placental ultrastructure, particularly the nature of the maternal-fetal interface.

Udgivelsesdato: 2007-Jul Placentation in tenrecs of the subfamily Oryzorictinae, family Tenrecidae, has not been described previously. The structure of the placenta of this group and especially of the genus Microgale was investigated to determine its similarity or dissimilarity to previously described placentas of the tenrec subfamilies Potamogalinae and Tenrecinae. Fifteen specimens of the genus Microgale ranging from an early yolk sac stage to near term were available for study. Placentation in Microgale was found to be different from other tenrecids in that there is an early simple lateral rather than central haemophagous region. In addition, a more villous portion of the placental disk forms before the formation of a more compact labyrinth. Although the definitive placenta is cellular haemomonochorial, it lacks the spongy zone found in the Tenrecinae. Neither does it resemble the endotheliochorial condition found in the Potamogalinae. Of the two genera of the subfamily Oryzorictinae represented by single specimens, the placenta of Limnogale resembled that of the Microgale but Oryzorictes had several differences including a lobulated placental disk. It is concluded that there is more variation in placentation both within the subfamily Oryzorictinae and within the family Tenrecidae than would ordinarily be expected

Udgivelsesdato: 2007-Apr

An overview is given of variations in placentation with particular focus on yolk sac, paraplacenta, and other structures important to histotrophic nutrition. The placenta proper varies in general shape, internal structure, and the number of tissues in the interhemal barrier. Yolk sac membranes persist to term in insectivores, colugos, rodents, and lagomorphs. In the latter two orders, they are of known importance for maternal-fetal transfer of antibodies, vitamins, lipids, and proteins. The detached yolk sac of bats is also active throughout gestation. A vascular paraplacenta, or smooth chorioallantois, has known functions in ruminants and carnivores and is found in several other orders of mammal where its function has yet to be explored. In human gestation, the chorion (avascular chorioallantois) is important for hormone synthesis. The true chorion of squirrels and hedgehogs is avascular but may nevertheless allow transfer from mother to fetus through the exocelom. Hemophagous areas with columnar trophoblast are paraplacental structures in carnivores and elephants but occur also within the placenta as in hyenas and moles. In shrews, it is the yolk sac that ingests and processes red cells. Areolas and chorionic vesicles are other structures important for absorption of uterine secretions and ingestion of cellular debris. In conclusion, we find that paraplacental structures, while showing less variation than the placenta proper, contribute not just to the integrity of overall placentation, but in various ways to maternal-fetal interrelationships.

We here review the evolution of invasive placentation in primates towards the deep penetration of the endometrium and its arteries in hominoids. The strepsirrhine primates (lemurs and lorises) have non-invasive, epitheliochorial placentation, although this is thought to be derived from a more invasive type. In haplorhine primates, there is differentiation of trophoblast at the blastocyst stage into syncytial and cellular trophoblast. Implantation involves syncytiotrophoblast that first removes the uterine epithelium then consolidates at the basal lamina before continuing into the stroma. In later stages of pregnancy, especially in Old World monkeys and apes, cytotrophoblast plays a greater role in the invasive process. Columns of trophoblast cells advance to the base of the implantation site where they spread out to form a cytotrophoblastic shell. In addition, cytotrophoblasts advance into the lumen of the spiral arteries. They are responsible for remodelling these vessels to form wide, low-resistance conduits. In human and great apes, there is additional invasion of the endometrium and its vessels by trophoblasts originating from the base of the anchoring villi. Deep trophoblast invasion that extends remodelling of the spiral arteries to segments in the inner myometrium evolved in the common ancestor of gorilla, chimp and human.

The present study determined whether estrogen plays a role in regulating invasion and remodeling of the uterine spiral arteries by extravillous trophoblasts during early baboon pregnancy. The level of trophoblast invasion of spiral arteries was assessed on day 60 of gestation (term is 184 days) in baboons untreated or treated on days 25-59 with estradiol or aromatizable androstenedione. The administration of estradiol or androstenedione increased (P0.01) maternal serum estradiol levels approximately 3-fold above normal. The mean+/-SE percentage of spiral arteries/arterioles invaded by extravillous cytotrophoblasts in estradiol-treated baboons for vessels with diameters of 26-50 microm (0.0+/-0.0), 51-100 microm (1.2+/-0.7) and100 microm (13.2+/-5.5) was 100%, 90%, and 75% lower (P0.001), respectively, than in untreated baboons (2.4+/-1.2%; 11.0+/-5.5%, and 54.5+/-8.5%, respectively). Similar results were obtained with androstenedione treatment. However, the distribution of uterine spiral arteries grouped by diameter or number of arteries per basal plate area, i.e. microvessel density, were similar in untreated and estrogen-treated baboons. We suggest, therefore, that the low levels of estrogen exhibited during early primate pregnancy are required to permit normal progression of trophoblast vascular invasion and that the surge in estrogen which occurs during the second-third of normal pregnancy has a physiological role in suppressing further arterial trophoblast invasion. Consequently, we propose that the estrogen-dependent restraint of spiral artery invasion/remodeling ensures optimal blood flow dynamics across the uteroplacental vascular bed to promote normal fetal growth and development.

BACKGROUND: Early in vitro studies of blastocyst formation in several primate species have demonstrated the feasibility of such studies. Initial studies of in vitro-fertilized oocytes cultured with buffalo rat liver cells suggested that other epithelial cells might be used to assess blastocyst adherence and penetration in vitro. METHODS: Macaque blastocysts were incubated with different epithelial cell lines or with Matrigel. The interaction was studied using light and transmission electron microscopy. RESULTS: In general, zona-free blastocysts attached 2 days after placing on the substrates. MDCK cells provided optimal conditions for blastocyst development. The best preparations showed some development of an amniotic cavity and distribution of cytotrophoblast and syncytial trophoblast. Distribution of syncytial trophoblast at the margin of the site and cytotrophoblast centrally was similar to that seen at the trophoblastic plate stage in this species. However, there was less syncytial trophoblast than is normally found at this stage, and total time from fertilization to the trophoblastic plate stage was delayed 2 days. CONCLUSIONS: While in vitro studies with blastocysts cannot completely mimic the intrauterine environment, they can illustrate some of the potential interactions and provide a situation in which parameters may be manipulated.

Afrotheria, one of four mammalian superorders, comprises elephants, sea cows, hyraxes, aardvark, elephant shrews, tenrecs and golden moles. Their placentas either form an equatorial band or are discoid in shape. The interhemal region, separating fetal and maternal blood, is endotheliochorial in elephants, aardvark and possibly the sea cows, but hemochorial in the remaining orders. There is a secondary epitheliochorial placenta in elephant shrews while a similar structure in tenrecs erodes maternal tissues. Specialized hemophagous regions are a striking characteristic of some of these placentas yet absent in hyraxes, elephant shrews, and golden moles. It is possible that the common ancestor of the Afrotheria had an endotheliochorial placenta. Establishment of a hemochorial condition, as seen in rock hyraxes, elephant shrews, tenrecs, and golden moles, would be a more recent development. The elephant, manatee, and aardvark all have circumferential placentas. Thus the formation of a discoid placenta with a more or less extensive secondary placenta in elephant shrews and tenrecs would also be a derived state.

Until recently, tenrecs were classified with insectivores in the order Lipotyphla, but nucleotide sequence data suggest they have closer affinities with a group of African mammals called Afrotheria. The placenta of Echinops has not been described and no studies involving electron microscopy of the placenta of any species of tenrec have been published. We used light and transmission electron microscopy to examine fixed placentae of embryos ranging from 25-66 mm in length. The placental disk is situated in the antimesometrial portion of the bicornuate uterus. The greater part of the disk consists of a labyrinth underlain by a spongy zone. The interhaemal barrier is unusual in that the trophoblastic component is a single layer of cytotrophoblast. These trophoblast cells have thick areas especially near the nuclei and extensive thin flanges but only occasionally have membrane-closed pore regions. The luminal surface has isolated patches of microvilli, and pinocytotic vesicles are numerous both apically and basally. In the centre of the placental disk is an elaborately folded haemophagous region. The primary folds have allantoic endoderm at one surface and columnar cytotrophoblast at the other. These trophoblast cells have numerous lipid droplets and vesicles, and often contain large yellow pigment crystalloids. The labyrinthine zone ends abruptly at the margins of the placental disk. However, the endoderm and connective tissue of the allantois and a layer of cytotrophoblast extend beyond the placental disk as a paraplacental region. Some of these distinctive features of Echinops placenta are shared with individual afrotherians, but no significant characteristic of definitive placentation is shared by all the Afrotheria.

BACKGROUND To ascertain if hepatic or renal dysfunction or prior pelvic radiation (XRT) leads to increased toxicity at a given dose of irinotecan and to characterize the pharmacokinetics of irinotecan and its major metabolites in patients with hepatic or renal dysfunction. PATIENTS AND METHODS Adults with tumors appropriate for irinotecan therapy and who had abnormal liver or renal function tests or had prior radiation to the pelvis were eligible. Patients were assigned to one of four treatment cohorts: I, aspartate aminotransferase (AST) > or = 3x upper limit of normal and direct bilirubin

In the course of a study on reproduction in the nine-banded armadillo, conceptuses between the beginning of implantation and primitive streak formation were examined to determine the manner of trophoblast differentiation during invasion of the endometrium and the sequence involved in formation of four identical quadruplets. The armadillo blastocyst implants in the fundic recess of the uterus. A single amnion and cup-shaped epiblastic plate are formed, and an exocelom develops between the amnion and trophoblast of the implantation site. Loss of the abembryonic trophoblast exposes both visceral and parietal endoderm to the uterine lumen, inverting the yolk sac. Continued expansion of the exocelom facilitates the intrusion of the forming conceptus into the uterine lumen and is accompanied by enlargement of the epiblastic plate. Separate areas of condensations of epiblast cells are the first indication of formation of the four identical quadruplets. The single layer of microvillous trophoblast with basal infoldings (designated absorptive trophoblast) is most likely to contribute extensively to movement of fluid into the exocelom. The resulting expansion of the exocelom not only enlarges the implantation site but also displaces the collapsing common amnion, limiting the amnion to the areas of the forming embryos.

The development of the baboon anchoring villus has been studied from day 14 to day 48 of gestation, using light and electron microscopy. At day 14, cords of trophoblast could be seen streaming into the endometrium, invading maternal vessels and forming blood-filled lacunae; by 20 days gestation some of these had differentiated into distinctive anchoring villi, with an outer covering of syncytiotrophoblast and inner cytotrophoblast cells which differed from those of floating villi in that a subpopulation detached from the syncytium to form an interconnecting network of cells within the centre of the villus. Subsequent migration of cytotrophoblast into the endometrium formed the cytotrophoblastic shell while fibrillin-like extracellular matrix biosynthesis within the body of the villus provided a firm mechanical support. At the trophoblast-decidual interface, a zone of necrosis and phagocytosis initially developed, which became less extensive with time, so that by 40 days a stable interface was evident with only residual pockets of necrosis. During this period, there was differentiation of decidual cells which by 28 days developed characteristic pedunculated cell processes, and later became surrounded by a basal lamina. The factors that control detachment of cytotrophoblast from the syncytium and the biosynthesis of the specialized, fibrillar extracellular matrix, features that are not apparent in other placental villi, require further investigation, possibly by carefully controlled in vitro experimentation.

Anchoring villi of first trimester placentae of the macaque, baboon and human were examined by light and electron microscopy. The anchoring villi of the baboon and macaque are similar in having more elongated cell columns than those of the human and in having more extracellular matrix between cytotrophoblast cells. These species also have a thicker and more uniform trophoblastic shell. The generative region of cytotrophoblast cells adjacent to the villous mesenchyme is similar in all three species, with the aspect of the core abutting this area being lined by a thickened basal lamina. Similarly, migratory cytotrophoblast cells form extracellular matrix in all three species, but matrix-rich regions of the anchoring villi and shell are more extensive in the non-human primates. The extracellular matrix and especially the material resembling fibrillin may serve to strengthen the villi, particularly the elongated villi of the non-human primate, and also may prevent maternal cells migrating into the trophoblastic shell. The baboon and macaque cytotrophoblast cells that form this matrix tend to be linked by gap and desmosomal junctions and are in contiguous arrays, whereas those in the human that are blocked from reaching normal decidua form abundant extracellular matrix but have no gap junctions. Whether the lack of extensive invasion of the endometrium by baboon and macaque cytotrophoblast cells is related to the increased amount of extracellular matrix, their greater distance from the mesenchymal core, or their intercellular linkages is not known. The investigation of isolated villi from the macaque or baboon, as has been extensively carried out in the human, might help to determine whether the cytotrophoblast cells are intrinsically different or are responding to different environmental cues.

The free allantois and allantochorion of conceptuses from 17 mares between 20 and 90 days of gestation were examined to determine the manner in which the associated mesodermal derivatives differentiated. It was found that a robust basement membrane developed under the allantoic endoderm, and that this basement membrane was partially isolated from the vascular layer of the allantois by a mesothelial layer and an exocelom-like space. The exocelom-like space persisted until approximately the stage of villous formation, and remnants of the space persisted over larger allantoic vessels even later. It is suggested that originally the presence of the mesothelial layer facilitates type I collagen formation in the thick basement membrane under the basal lamina of the allantoic endoderm. The basement membrane maintains the integrity of the allantois while the underlying space allows some slippage between the allantois and its vascular layer which is more closely associated with the endometrial and exocelomic surfaces, respectively.

The most profound changes in the relationship of the trophoblast to endometrial tissues occur in the first 5 days after the initiation of implantation. Not only have the earliest stages--adhesion and epithelial penetration--never been seen in the human, but also the trophoblastic plate and lacunar stages that follow are not available for modern investigative methods. Studies of appropriately timed endometrium and of trophoblast- and endometrium-derived cell lines have important implications for aspects of implantation. Use of nonhuman primates and other animal models for appropriate stages in implantation could further our understanding of direct trophoblast-uterine interactions. The mechanisms involved in epithelial penetration by infiltration of the syncytial trophoblast into the uterine luminal epithelium could be studied profitably using the marmoset or the ferret. Drawing of the blastocyst into an interstitial location might be investigated in the guinea pig. Formation of trophoblastic lacunae can be investigated in the cynomolgus monkey. By using such animal models of events of implantation in situ, implications concerning the molecules involved in adhesion, penetration of junctional complexes, and uterine vessel invasion that have been derived from in vitro or murine studies may be placed in context.

The hypothesis that fibrin type fibrinoid deposition on villi is unique to the term human placenta was tested. Bright-field microscopy was used to examine sections of first and second trimester human placental villi and tissues from three animal species that have villous haemochorial placentae similar to the human: the armadillo, the baboon and the rhesus. Sections stained with haematoxylin and eosin showed fibrin type fibrinoid deposits were hypocellular, eosinophilic masses attached to the surface of villi examined from both the human and the animal specimens. The deposits were located at discontinuities in the syncytiotrophoblast layer, and the fibrinoid provided a matrix for trophoblast re-epithelialization of the villous surface. It is concluded that fibrin-type fibrinoid is not unique to the term human placenta. The presence of the syncytiotrophoblast discontinuities associated with the fibrinoid deposition must be considered in models of maternal-fetal exchange in the villous haemochorial placenta.

Introduction The Laotian rock rat is a relict species in a sister group relationship to hystricognath rodents (Hystricognathi). We asked whether there were similarities in placentation that might reflect this relationship or differences that might cast light on the evolution of Hystricognathi. Methods We examined the reproductive tract of nonpregnant ( n = 5), early ( n = 3) and mid to late gestation ( n = 2) females. Selected characters were mapped to a phylogenetic tree to examine their evolution in rodents. Results The chorionic placenta was discoid and labyrinthine with a spongy zone but without internal lobes. The interhemal region was hemodichorial with syncytiotrophoblast lining maternal blood spaces and an inner layer of vacuolated cytotrophoblast. There was no subplacenta. The yolk sac was well developed with a villous portion that faced the placental disk but no fibrovascular ring. There was a single fetus that very likely would be precocial at birth. Discussion A lobulated labyrinth and the presence of a subplacenta and a fibrovascular ring emerged as synapomorphies for Hystricognathi. Laonastes , Ctenodactylus and stem Hystricognathi all had precocial young, whereas altriciality was the plesiomorphic condition for rodents. A hemomonochorial interhemal region was plesiomorphic for rodents and Hystricognathi, and the hemodichorial condition found in Laonastes , and possibly in Ctenodactylus , was unlike that of any rodent studied to date. Conclusion Similar to Hystricognathi, Laonastes bears precocial young, but this species lacks placental adaptations such as the subplacenta, suggesting they were evolved subsequent to a change in reproductive strategy in the common ancestor of Laonastes and Hystricognathi.

Epitheliochorial placentation is a derived condition and has evolved separately in strepsirrhine primates and laurasiatherians (pangolins, whales, and hoofed mammals). Usually it is associated with a long gestation period, small litters, and precocial young. Oxygen transfer is facilitated by indenting of the uterine and trophoblast epithelia by maternal and fetal capillaries, respectively. Histotrophic nutrition is important, and adaptations include areolas and hemophagous regions. In pigs and horses, for example, iron is transported as uteroferrin secreted from the uterine glands and taken up by areolas. In the horse, invasive trophoblast cells form cups within the endometrium that are the source of equine chorionic gonadotropin. In ruminants, binucleate trophoblast cells fuse with uterine epithelial cells to form trinucleate cells or plaques that secrete pregnancy hormones. There is evidence of immunosuppression in connection with these more invasive types of trophoblasts. The epitheliochorial condition may be advantageous for long pregnancies in large animals.

Adhesion of trophoblast of the blastocyst to and penetration into the uterine epithelium, invasion into the maternal vessels and endometrial stroma, and establishment of the basic organization of the placenta all occur within the first week following the initiation of implantation in the human, macaques and several other primates. The cellular rearrangements and interactions of trophoblast with endometrial epithelial cells and stroma were studied during this periimplantation stage in macaques. At the early lacunar stage, 1–2 days after the initiation of implantation, both cytotrophoblast and syncytial trophoblast can be found at the maternal surface of lacunae. As the lacunar stage advances, both syncytial trophoblast and cytotrophoblast are found throughout the implantation site including in septae partitioning lacunae, but syncytial trophoblast lines most of the lacunae and forms the confluence with the maternal vessels. Indentations of fetal mesenchyme and accumulation of cytotrophoblast cells within the septae occur rapidly. Over a period of about 2 days, clusters of cytotrophoblast cells pass beyond the syncytial trophoblast at the maternal surface as the anchoring villi, establishing the trophoblastic shell. The bypassing of epithelial plaque cells by cytotrophoblast cells and elimination of stromal matrix between such clusters verifies that this is a progressive invasion rather than simply superficial growth. Concomitant with establishment of the trophoblastic shell there is extravasation of blood, necrosis of plaque cells, and massive invasion of vessels by cytotrophoblast resulting in a necrotic zone between the trophoblastic shell and endometrium. It is concluded that only in the brief period of time of establishment of the trophoblastic shell is the full invasive potential of cytotrophoblast realized and that only at this stage does cytotrophoblast demonstrate the type of invasive and migratory behavior which has been achieved with isolated human cytotrophoblast cells in vitro.

The distribution of cytokeratin-positive interstitial trophoblast cells in the endometrium of the macaque during placental development was examined. Such cells are moderately abundant only from the 15th through the 22th day of pregnancy, although there is considerable individual variation. During this period of gestation, interstitial trophoblast cells are distributed in the perivascular stroma immediately surrounding spiral arteries, including coils of arteries already invaded by endovascular trophoblast. The interstitial trophoblast cells are not seen to directly intrude into the smooth muscle of the spiral arteries. Very few interstitial trophoblast cells are present from days 12 through 14 of gestation when the arteries are first invaded by endovascular trophoblast. Even fewer interstitial trophoblast cells are seen after day 30 of gestation. The brief time in gestation when interstitial trophoblast cells are abundant coincides with a time when spiral arteries are undergoing rapid modification into patent uteroplacental arteries. It is suggested that during this period, the interstitial trophoblast cells may facilitate changes in shape of the spiral arteries by alterations of the perivascular connective tissue, perhaps through interactions with other cellular constituents rather than by directly modifying the arteries per se.

International audience; Workshops are an important part of the IFPA annual meeting as they allow for discussion ofspecialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which aresummarized in this report. These workshops related to various aspects of placental biology: 1)immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stemcells.

A rat IgM monoclonal antibody has been developed which recognized a mouse germ cell nuclear antigen (GCNA1). GCNA1 is present in prospermatogonia (gonocytes) in males and in oogonia and oocytes of females within the gonadal ridge from Embryonic Day 11.5 onward, but rarely in primordial germ cells prior to their arrival at the gonadal ridge. Immunolocalization demonstrates that GCNA1 is abundant in nuclei of spermatogonia and early spermatocytes, but decreases during subsequent spermatocyte and round spermatid development, and is not detected beyond step 10 elongating spermatids. The antigen is approximately 80-110 kDa on immunoblots of isolated pachytene spermatocytes and round spermatids. However, GCNA1 appears to be absent from sperm in the epididymis and vas deferens, Sertoli cells, TM3 cells (Leydig-like) and TM4 cells (Sertoli-like), lung, liver, kidney, spleen, heart, skin, brain, epididymis, and ovary. GCNA1 is present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in prepuberal male mice (Days 2-14) in all stages of prespermatogonial and spermatogonial development. It is also present in oocytes of neonatal females until Postpartum Day 12. GCNA1 is first lost from oocytes in the medulla of the ovary as they arrest at the dictyate stage and gain a layer of granulosa cells. In addition, antigen is present in moderate amounts in F9 embryonal carcinoma cells and SCC-PSA1 pluripotent terato-carcinoma cells. Thus, GCNA1 serves as a common marker of the germ cell lineage in male and female mice after primordial germ cells arrive in the gonadal ridge until they reach the diplotene/dictyate stage of the first meiotic division.

An individual embryo from day 15 of gestation in the rhesus monkey showed partial separation of the cranial end of the embryonic shield into two separate cell masses with separate amnions but with a common yolk sac. Although what the fate of this conceptus might have been is not known, it demonstrates that the physical features of implantation in this primate are permissive of relatively late division of the epiblast, which suggests that monozygotic twinning may occur as late as the embryonic shield stage just prior to primitive streak formation.

Endotheliochorial placentas occur in orders from all four major clades of eutherian mammal. Species with this type of placenta include one of the smallest (pygmy shrew) and largest (African elephant) land mammals. The endotheliochorial placenta as a definitive form has an interhemal area consisting of maternal endothelium, interstitial lamina, trophoblast, individual or conjoint basal laminas, and fetal endothelium. We commonly think of such placentas as having hypertrophied maternal endothelium with abundant rough endoplasmic reticulum (rER), and as having hemophagous regions. Considering them as a whole, the trophoblast may be syncytial or cellular, fenestrated or nonfenestrated, and there may or may not be hemophagous regions. Variations also appear in the extent of hypertrophy of the maternal endothelium and in the abundance of rER in these cells. This combination of traits and a few other features produces many morphological variants. In addition to endotheliochorial as a definitive condition, a transitory endotheliochorial condition may appear in the course of forming a hemochorial placenta. In some emballonurid bats the early endotheliochorial placenta has two layers of trophoblast, but the definitive placenta lacks an outer syncytial trophoblast layer. In mollosid bats a well developed endotheliochorial placenta is present for a short time even after a definitive hemochorial placenta has developed in a different region. It is concluded that the endotheliochorial placenta is more widespread and diversified than originally thought, with the variant with cellular trophoblast in particular appearing in several species studied recently.

It is argued that the successful placental arrangements are those that decrease the exposure of the trophoblast to the efferent side of the cellular immune response. Examples are taken from three different groups with hemochorial placentation, to show that their placental morphology places most of the trophoblast in contact with maternal blood, not maternal connective tissue. In addition, maternal cells of the junctional area are modified either before contact with trophoblast (rat) or just after trophoblast invasion (primate), or the region of contact is limited as in the armadillo, in which maternal blood sinuses are expanded to form intervillous spaces.

The processes by which trophoblast cells invade and modify the walls of the uteroplacental arteries of macaques during the course of gestation were examined. Antibodies to cytokeratins were employed to identify trophoblast, anti-desmin antibody to identify smooth muscle, and antibodies to type IV collagen, laminin, and fibronectin to examine changes in extracellular matrix distribution in the arterial wall. During early gestation, endovascular trophoblast adhered to the arterial wall, often in an asymmetrical distribution. As trophoblast cells moved outwardly into the tunica media, the basement membrane underlying the endothelium was lost, as indicated by gaps in the layer when stained for type IV collagen and laminin. Trophoblast cells became sequestered in the vessel wall where they hypertrophied and became surrounded by a capsule containing type IV collagen and laminin. As the trophoblast cells became established in the vessel wall, the muscular layer of the artery became discontinuous. Throughout gestation it was common for trophoblast cells to invade the vessel intimal layer and share the lining of the artery with typical endothelial cells. This general disposition of endovascular and intramural trophoblast persisted into late gestation. In addition, and contrary to the results of earlier studies of macaques, we identified trophoblastic invasion and modification of myometrial segments of the uteroplacental arteries in later gestation. We also found evidence of interstitial trophoblast cells among the stromal cells of the endometrium, especially during early gestation.

Uterotubal junction tissues (n=12) recovered from young nulliparous mares 4 h after insemination were examined for spermatozoa in situ using scanning electron microscopy. Mares were inseminated before ovulation with semen containing 66-85% morphologically abnormal spermatozoa and approximately 1 x 10(9) progressively motile spermatozoa. Spermatozoa were found in all the tissues and displayed a variety of relationships to the epithelium, depending on regional location, spatial constraints and epithelial surface characteristics. Spermatozoa were most abundant in the epithelial folds on the uterine side of the uterotubal junction papilla. More than 90% of the spermatozoa had normal morphology. These observations indicate that morphologically normal equine spermatozoa develop specific associations with the luminal epithelium of the reproductive tract during transport to the oviduct. Furthermore, the uterine papilla of the uterotubal junction may be a site of preovulatory sperm storage. The scarcity of morphologically abnormal spermatozoa, despite their abundance in the inseminate, indicates that most abnormal spermatozoa either do not reach the uterotubal junction or do not develop normal associations with the epithelium of the uterotubal junction. This observation provides evidence for a mechanism of sperm selection during transport. Hence, both normal morphology and progressive motility of spermatozoa should be considered in determining insemination doses, particularly for subfertile stallions.

Udgivelsesdato: 2009-Oct-14 Interest in insectivoran grade mammals has been reawakened by taxonomic changes that place tenrecs and golden moles in a new order and separate hedgehogs from moles, shrews and solenodons. This survey of their placentation shows there is great variation even within families. As an example three subfamilies of tenrec have been examined. The interhemal region is cellular hemomonochorial in Echinops and Microgale but endotheliochorial in Micropotamogale. Golden moles, which are placed in the same order, have hemodichorial placentation. Many insectivores have complex arrangements for histotrophic nutrition involving columnar trophoblast cells. These range from areolae in moles through complexly folded hemophagous regions in tenrecs to the trophoblastic annulus in shrews. Of these placental characters, few offer support to current phylogenies. However, the case for placing hedgehogs and gymnures in a separate order (Erinaceomorpha) is bolstered by the presence of interstitial implantation, amniogenesis by cavitation, a hemochorial barrier and a prominent spongy zone; these features do not occur in shrews, moles or solenodons (Soricomorpha). Three insectivoran grade mammals deserve close attention as they have been selected for genome sequencing. One of these, the European hedgehog (Erinaceus europaeus), has not been studied with current methodology and renewed investigation of this or the closely related genus Atelerix should be a priority.

Background Sperm desiccation is an attractive approach for sperm preservation. In this study, we examined the feasibility and efficiency of intracytoplasmic sperm injection using vacuum-dried rhesus macaque sperm in CZB medium supplemented with 10% fetal bovine serum. Methods A total of 109 MII oocytes were injected with 69 fresh ejaculated sperm and 40 vacuum-dried sperm. Results Cleavage occurred in 97% of oocytes injected with fresh, motile sperm and in 88% of oocytes injected with vacuum-dried sperm. Of the cleaved oocytes, 68% fresh sperm-injected oocytes and 74% of dried sperm-injected oocytes developed to the compact morula stage. Blastocyst development was comparable between fresh-injected (16%) and vacuum-dried-injected (17%) oocytes. Differences between treatment groups were not significant. Transmission electron microscopic observation of the blastocysts indicated no detectable differences between fresh sperm and dried sperm-derived embryos. Conclusions We conclude that vacuum-dried rhesus macaque sperm are capable of inducing fertilization and development of pre-implantation embryos when sperm were dried under vacuum and microinjected into normal viable oocytes.

The structure of the equine chorionic girdle between days 28 and 42 of gestation was examined. Of particular interest were differentiation of trophoblastic cells within the girdle, adhesion between girdle and endometrium, invasion and displacement of the uterine epithelium, and the nature of the endometrium when girdle cells migrate into it to form endometrial cup cells. The chorionic girdle, identified initially as a band of tall columnar cells, becomes a stratified columnar epithelium indented by clefts and pits. Adhesion to and penetration through the endometrial luminal epithelium are rapid and occur initially in very limited areas. Stromal invasion occurs as strands of contiguous trophoblast cells invade through the basal lamina. Only girdle cells that are adjacent to the basal lamina or have entered the endometrial stroma undergo hypertrophy and differentiate into cup cells. At the initiation of trophoblastic invasion, the luminal epithelium contains numerous, large, intra-epithelial, granular lymphocytes; small lymphocytes then accumulate in the stroma, but by day 42 lymphocytes are largely confined to the periphery of the cup. Although adhesion of trophoblast to the endometrial surface is initiated by small groups of girdle cells on restricted areas of the endometrial folds, the area is then increased by new areas of adhesion and by expansion of the initial invasion. Areas of girdle cells that do not attach undergo necrosis, as do superficial portions of areas of invasion. Consequently the girdle cells that form cups may be a minority of the original population. It is suggested that the differentiation of girdle cells is closely programmed and that cells that do not reach the stroma become necrotic at the same time that endometrial cup cells are differentiating.

Many early embryonic stages are nearly indistinguishable in different Eutheria. However, implantation stages and placental morphological types vary tremendously. A number of factors favor this conflicting diversity. 1. Whereas embryo development takes place in the isolation of the amniotic cavity, the extraembryonic membranes of the conceptus develop in close association with the uterus of a genetically different organism. 2. Early conditions for the developing conceptus are anaerobic whereas later in development efficient aerobic conditions are essential for continued growth of the fetus. 3. Developing extraembryonic membranes have the potential to form two partially sequential placentas. The yolk sac can participate in forming a choriovitelline placenta, including an interhemal region, and can be adapted to various non-respiratory functions as gestation proceeds. Development of the chorioallantoic placenta begins later than the choriovitelline placenta but can overlap with this before supplanting it. The original development of the extraembryonic membranes occurs when the conceptus is sufficiently small that neither its nutritional requirements nor its respiratory needs are the burden to the maternal organism that they are later in gestation. Despite these developmental factors promoting different methods of forming the definitive placenta, the placental type is consistent within most families indicating that the divergence in placental structure accompanied evolution of differences between groups.

We have shown that advancing the increase in maternal serum estrogen levels from the second to the first third of baboon pregnancy suppressed extravillous cytotrophoblast (EVT) spiral artery invasion. Because vascular endothelial growth factor (VEGF) promotes EVT invasion, the present study determined whether EVT VEGF expression is altered by prematurely elevating estrogen in early pregnancy. Placental basal plate was obtained on d 60 of gestation (term is 184 d) from baboons treated daily on d 25–59 with estradiol (0.35 mg/d sc), which increased maternal peripheral serum estradiol levels 3-fold above normal. Overall percentage of uterine arteries (25 to more than 100 μm in diameter) invaded by EVT assessed by image analysis in untreated baboons (29.11 ± 5.78%) was decreased 4.5-fold (P < 0.001) by prematurely elevating estrogen (6.55 ± 1.83%). VEGF mRNA levels in EVT isolated by laser capture microdissection from the anchoring villi of untreated baboons (6.77 ± 2.20) were decreased approximately 5-fold (P < 0.05, ANOVA) by estradiol (1.37 ± 0.29). Uterine vein serum levels of the truncated soluble fms-like receptor, which controls VEGF bioavailability, in untreated baboons (403 ± 37 pg/ml) were increased 3-fold (P < 0.01) by estrogen treatment (1127 ± 197 pg/ml). Thus, placental EVT expression of VEGF mRNA was decreased and serum soluble truncated fms-like receptor levels increased in baboons in which EVT invasion of the uterine spiral arteries was suppressed by advancing the rise in estrogen from the second to the first third of pregnancy. We suggest that VEGF mediates the decline in EVT vessel invasion induced by estrogen in early primate pregnancy.

Hemomonochorial placentation occurs in diverse species. We have examined placental glycosylation in five widely separated mammals with this type of placentation – lesser hedgehog tenrec (Echinops telfairi), spotted hyena (Crocuta crocuta), nine-banded armadillo (Dasypus novemcinctus), human (Homo sapiens) and guinea pig (Cavia porcellus) – in order to assess whether evolutionary convergence to the hemomonochorial state is accompanied by a similar convergence of glycan expression. Placentae from 2 E. telfairi, 3 C. crocuta, 1 D. novemcinctus, 4 womenand 1 C. porcellus were fixed and processed into epoxy resin. Binding of twenty-three lectins was assessed using a semiquantitative ranking system. The trophoblast apical/microvillous membrane of all five species showed marked similarities in glycosylation. In the N-linked series, there were abundant bi/tri-antennary complex chains, while the non-bisected variants were much scarcer. All species had plentiful N-acetyl lactosamine sequences; at chain termini, binding to Galβ1,4GlcNAc and Galβ1,3GalNAc sequences was greatly enhanced after neuraminidase treatment. In all species, terminal NeuNAcα2,3 residues were detected. The tenrec had unusually abundant terminal N-acetyl galactosamine. The basal plasma membrane/basal lamina showed glycosylation patterns distinct from the microvillous membrane in each case, indicating chemical diversity of the two opposite faces of trophoblast. Similar classes of glycan at the hemochorial interface suggest conservation of function. The observed lectin binding patterns suggest broad similarities of glycosylation that may have arisen by convergent evolution.

The hyena placenta is unique among carnivores in being hemochorial. It also has areas of erythrocyte uptake that differ from those seen in more commonly studied carnivores. The availability of timed midterm pregnancies made it possible to examine the organization of the placenta, the distribution of regions of columnar trophoblast and the nature of the heterophagous and hemophagous regions in well-preserved material. The labyrinth of the placenta is orderly arranged with periodic primary villi that have a surface of syncytial trophoblast. Secondary projections from the primary villi anastomose extensively. The junctional extreme of each primary villus forms an expanded tip covered by columnar trophoblast cells. Marginal to the hemochorial placenta, the paraplacenta is exposed to varying amounts of extravasated maternal blood which the cytotrophoblast cells ingest and destroy. Columnar trophoblast cells at the tips of the primary villi, in the villi of paraplacental margins of the labyrinth, in isolated patches within the labyrinth and beneath the allantochorionic plate are all capable of and are variably involved in erythrophagocytosis. Variations in the structure and the lectin histochemistry of the columnar trophoblast cells indicate variation depending on the exposure of these cells to erythrocytes and/or other histotrophic materials. The widespread distribution of the hemophagous regions suggests a nutritive function in addition to an iron transfer function of the columnar cytotrophoblast.

Udgivelsesdato: april 2006 In considering the diversity of Eutherian mammalian placental structure, it is helpful to keep in mind that both phylogenetically and ontogenetically a functional yolk sac placenta precedes development of the chorioallantoic placenta. Usually the chorioallantoic placenta progressively displaces the area of contact of the yolk sac placenta with the endometrium. It is also closely applied to the endometrium, increasing respiratory efficiency but tending to decrease histotrophic nutrition. Catarrhine primates have minimal histotrophic uptake, using haemotrophic mechanisms throughout most of gestation. Rodents, by using partial or complete inversion of the yolk sac, have extensive regions available to histotroph in later pregnancy. Most mammals, however, have developed specialized regions of the chorioallantoic placenta for ingestion of uterine secretions, cell debris and erythrocytes. These regions vary from simple saccular areolae to more complex areolae such as the chorionic vesicles of prosimian primates to specialized haemophagous areas. All such structures are consistently lined by columnar cellular trophoblast. Examples of these structures, many of which can ingest both glandular secretions and erythrocytes (i.e. they are heterophagous), can be found in species whose definitive placenta is epitheliochorial. They are common but not universal in species whose definitive placenta is endotheliochorial and are even found in a few species with definitive haemochorial placentas. Restriction of phagocytosis to regions of columnar cells provides polarized cells appropriately oriented for ingestion, breakdown and transport, and limits the type of ingestion of both beneficial and potentially toxic materials to expendable individual cells.

The otter shrews are members of the subfamily Potamogalinae within the family Tenrecidae. No description of the ovaries of any member of this subfamily has been published previously. The lesser hedgehog tenrec, Echinops telfairi, is a member of the subfamily Tenrecinae of the same family and, although its ovaries have not been described, other members of this subfamily have been shown to have ovaries with non-antral follicles. Examination of these two species illustrated that non-antral follicles were characteristic of the ovaries of both species, as was clefting and lobulation of the ovaries. Juvenile otter shrews range from those with only small follicles in the cortex to those with 300- to 400-microm follicles similar to those seen in non-pregnant and pregnant adults. As in other species, most of the growth of the oocyte occurred when follicles had one to two layers of granulosa cells. When larger follicles became atretic in the Nimba otter shrew, hypertrophy of the theca interna produced nodules of glandular interstitial tissue. In the tenrec, the hypertrophying theca interna cells in most large follicles appeared to undergo degeneration. Both species had some follicular fluid in the intercellular spaces between the more peripheral granulosa cells. It is suggested that this fluid could aid in separation of the cumulus from the remaining granulosa at ovulation. The protruding follicles in lobules and absence of a tunica albuginea might also facilitate ovulation of non-antral follicles. Ovaries with a thin-absent tunica albuginea and follicles with small-absent antra are widespread within both the Eulipotyphla and in the Afrosoricida, suggesting that such features may represent a primitive condition in ovarian development. Lobulated and deeply crypted ovaries are found in both groups but are not as common in the Eulipotyphla making inclusion of this feature as primitive more speculative.

BACKGROUND: Loss of the transmembrane mucin, Muc-1, is a molecular correlate of the acquisition of uterine receptivity to embryo adhesion in most species examined. In macaques, two distinct adhesion events occur at opposite sides of the uterus. Attachment to the secondary site is delayed relative to the primary site. The aim was to determine if Muc-1 is removed at secondary sites prior to trophoblast attachment. METHODS: We examined Muc-1 expression in the uteri of cynomolgus macaque and baboon during early implantation by immunocytochemistry. RESULTS: Luminal epithelia were devoid of Muc-1 at all stages examined at both primary and secondary adhesion sites. Loss of Muc-1 in luminal epithelia was found to be maternally determined, accompanied membrane transformation in both macaque and baboon, and at secondary implantation sites, preceded trophoblast attachment. In contrast, glandular epithelia in pregnant macaques exhibited a temporal and compartmentalized gradient of Muc-1 loss confined to the implantation sites. Glandular epithelia in the pregnant baboon uterus were uniformly negative for Muc-1. CONCLUSIONS: Restriction of the Muc-1 loss in glandular epithelia to conceptual cycles may reflect the fundamental distinctions among epithelia of the various uterine compartments and the differential modulation of Muc-1 that occurs within these compartments in conceptual and non-conceptual cycles.

The otter shrews of mainland Africa are the closest relatives of the Madagascar tenrecs. We sought for similarities in placentation between the two groups and, in a wider context, with other mammals of the Afrotheria clade. Specimens of the Nimba otter shrew (Micropotamogale lamottei) were obtained from the Ivory Coast and examples of the giant otter shrew (Potamogale velox) from the Hill Collection. The Nimba otter shrew has a central haemophagous organ similar to that in tenrecs. The labyrinth of the Nimba otter shrew, however, is endotheliochorial with syncytial trophoblast enclosing the maternal vessels. On the other hand tenrecs have cellular haemomonochorial placentae and an associated spongy zone, which is not present in the Nimba otter shrew. The placenta of the giant otter shrew is also endotheliochorial. The central region of its placenta is particularly interesting, since the juxtafetal portion is clearly a haemophagous region whereas the labyrinth feeding this region is endotheliochorial. Thus there is considerable variation in placental morphology within Tenrecidae. Importantly, however, both otter shrews have a large allantoic sac divided into four intercommunicating lobes by two pairs of septal folds. A similar arrangement has been described for representatives of each of the remaining five orders within Afrotheria. This is significant because previous anatomical studies have failed to establish a single synapomorphy in support of Afrotheria.

Our purpose was to determine how the central haemophagous region and cellular haemomonochorial labyrinth of the tenrec placenta are formed. The haemophagous region is preceded by a region of invasion of the endometrium by trophoblast comprising a cytotrophoblast layer covered by syncytial trophoblast and contiguous with numerous masses of multinucleate trophoblast. The trophoblast intrudes into the endometrium, eliminating the stroma, although small vessels and clumps of glandular epithelium persist. This extensive central region is connected to the forming disk by a ring of chorioallantois covered by a single layer of columnar trophoblast. Later the multinucleate masses and syncytial trophoblast degenerate. The unilaminar cytotrophoblast remains, is elaborated into folds, and phagocytoses glandular secretion, cell debris and erythrocytes. As the central area is transforming, fetal capillaries move into the cytotrophoblast pads surrounding the central zone. Prior to this, the cytotrophoblast has formed a multilayered structure and interrupted maternal vessels to create an anastomotic network of blood spaces lined by cytotrophoblast. The invasion of fetal capillaries transforms this preplacental pad into a cellular haemomonochorial labyrinth with the uninvaded portion forming an underlying spongy zone. Thus interaction of the trophoblast with the endometrium is substantially different in the central zone compared to the area of the preplacental pad.

The diversity of placental structures in Eutherian mammals is such that drawing generalizations from the definitive forms is problematic. There are always areas of reduced interhaemal distance whether the placenta is epitheliochorial, synepitheliochorial, endotheliochorial or haemochorial. However, the thinning may be achieved by different means. The presence of a haemophagous area as an iron transport facilitator is generally associated with endotheliochorial placentae but is also found in sheep and goats (synepitheliochorial) and in tenrecs and hyaenas (haemochorial). Although similar chorioallantoic placentae are found within families, structure begins to diverge at the ordinal level and there is little correlation at the supraordinal level of phylogeny. Differences in formation and function of the yolk sac provide additional variation. There would appear to be considerable adaptive pressure for development or retention of the haemochorial type of chorioallantoic placenta. This type of placenta has several possible drawbacks including more ready passage of fetal cells to the maternal organism and, should the haemochorial condition be achieved early, oxidative stress. At any rate no animal larger than the human and gorilla has this type of placenta. The endotheliochorial condition is found in animals as large as the bears, manatee and elephants. In addition to the ungulates, the epitheliochorial condition is present in the largest animals with the longest gestation periods, the whales. Considering the length of time since the early stages of mammalian evolution, it is probable that few unmodified structural features are present in any currently surviving mammal. Nevertheless, more complete studies of divergent types of mammalian placenta should help our understanding of mammalian interrelationships as well as placental function.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3betaHSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc. In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development.

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor alpha (IL-11Ralpha). The present study examined immunoreactive IL-11 and IL-11Ralpha in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Ralpha increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Ralpha was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Ralpha was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.

The purpose of the study was to verify the knowledge of male university students regarding induced abortion. The sample was comprised of 71 male students that corresponded to 70% of the population made up of students from different health related programs at a federal university. Data was collected in August and October of 1998, by means of a questionnaire that was based on two previous exploratory studies. The respondents were 17 to 22 years old, catholic, single and financially dependent. The results demonstrated that a large percentage of male students had knowledge of what an induced abortion was and of its consequences, as well as of the methods used to induce the fetal loss. The respondents expressed a negative opinion about abortion and placed the responsibility for such a decision on the couple. They also indicated that besides the woman, other individuals, such as friends and family members bared some responsibility for the decision to end or not a pregnancy.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

This study was initiated to examine the early stages of trophoblast adhesion and invasion during implantation in the marmoset. Seven implantation sites were found in the uteri of four marmosets taken between days 13 and 15 of gestation. Three implantation sites in two uteri were examined in detail by electron microscopy. Between days 13 and 15, the marmoset implantation site expanded peripherally by adding areas where syncytial trophoblast penetrated between uterine luminal epithelial cells. Such penetrating masses often bridged openings of endometrial glands, shared junctional complexes with the uterine epithelial cells between which they are infiltrating, and subsequently reached the residual basal lamina of the uterine luminal epithelium. Centripetal to the peripheral region was an intermediate region in which syncytial trophoblast overlay individual clusters of epithelial cells and rested along the basal lamina. In this region there was some evidence of fusion of syncytial trophoblast with uterine epithelial cells. In the central region of the implantation site near the inner cell mass and amnion the trophoblast formed elaborate lamellipodia in relation to the basal lamina. In one of the three specimens examined with electron microscopy there were two foci where trophoblast penetrated through the basal lamina. It was also in the central region that trophoblast penetrated farthest into the uterine glands. The gland cells closest to trophoblast were less closely associated and lost their columnar shape, forming large round cells similar to the epithelial plaque cells of other primates. Where two blastocysts implanted on the same side of the uterus a conjoint membrane was formed which in regions consisted solely of syncytial trophoblast with two basal surfaces and two basal laminas. The prolonged period of time when the implantation site expands within the plane of the uterine epithelium (trophoblastic plate stage) and the peripheral to central sequence in extent of development make this primate a particularly useful animal for studies of trophoblast adhesion to and penetration of the uterine luminal epithelium.

Using lectin histochemistry on plastic-embedded material, the glycosylation patterns of equine girdle and cup cells, and associated endometrial glands, have been investigated from 37 to 67 days gestation. Results were compared with the glycosylation of the 50-day allantochorionic trophoblast of the established equine placenta that will later form the microcotyledons. The differentiated cup cells, which secrete equine chorionic gonadotropin (eCG), showed a pattern of glycosylation that was distinct both from the progenitor girdle cells and the allantochorionic trophoblast, with granules that bound lectins indicating high levels of α2,6 and α2,3-linked sialic acid, N-acetyllactosamine and bi/tri antennary non-bisected and bisected complex N-glycan. This is consistent with the known carbohydrate content of eCG. In contrast, the allantochorionic trophoblast at 50 days lacked detectable amounts of sialic acid and showed high levels of tri/tetra-antennary non-bisected complex N-glycan and N-acetyl galactosamine which was absent in the cup cells. During the process of girdle cell migration into maternal tissues, the uterine glands became greatly enlarged and dilated basally, with increased amounts of glycosylated secretory products revealed by lectins, which often seeped out into the extracellular space via ruptures in the apical regions of the gland wall.