Linda Romagnano

Egg removal and intraspecific brood parasitism in the European starling (Sturnus vulgaris).

SummaryFrom 1983 to 1986 we monitored 284 European starling (Sturnus vulgaris) nests in New Jersey for evidence of intraspecific brood parasitism and egg removal during the laying period. Egg removal occurred significantly more often at nests where intraspecific brood parasitism was detected (12 of 35 nests, 34%) than at unparasitized nests (23 of 249 nests, 9%). Brood parasitism (92% of parasitized nests) and egg removal (74% of nests with egg removal) were most common at nests where egg laying began in April of each year (i.e., early nests). Egg removal occurred at 26 (19%) and brood parasitism at 32 (23%) of 138 early nests. Both brood parasitism and egg removal were concentrated during the first four days in the laying period when brood parasitism is most likely to be successful and when host nests are most vulnerable to parasitism (Romagnano 1987). Both parasitism and removal usually involved a single egg at each nest. We detected brood parasitism and egg removal on the same day at five of 12 nests (42%) where both were observed. Because starlings do not remove foreign eggs from their nests once they begin laying (Stouffer et al. 1987) we hypothesize that parasite females sometimes removed host eggs while parasitizing nests.

Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro

Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracelluar digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast‐conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro‐enzymes. Dev Dyn 1999;216:374–384. ©1999 Wiley‐Liss, Inc.

The role of murine cell surface galactosyltransferase in trophoblast: Laminin interactions in vitro☆

Implantation of the mouse embryo involves the invasion of the secondary trophoblast giant cells of the ectoplacental cone (EPC) into the uterine decidua. The mechanisms of this event are poorly understood. The putative substrate molecules found in the decidua which could support trophoblast invasion include laminin, fibronectin, and collagen type IV. EPCs dissected from Day 7.5 embryos were cultured on all three matrices. Galactosyltransferase (GalTase) was localized by immunolabeling on trophoblast cell surfaces when grown on laminin but not the other matrices. Perturbations of the enzyme:substrate complex with alpha-lactalbumin, uridine diphosphogalactose, anti-GalTase, and pregalactosylation of the matrix did not affect rates of EPC attachment. However, decreased rates of migration or altered morphologies of spreading cells were observed. Laminin, and not fibronectin or collagen type IV, could be galactosylated with both exogenous GalTase or EPC outgrowths. Digests of galactosylated laminin produced a glycoconjugate substrate with a molecular weight of less than 10K. The results suggest that invasive secondary trophoblast cells possess a GalTase-mediated migration system that is functional on laminin.

Localization and expression of fibronectin during mouse decidualization in vitro: Mechanisms of cell:matrix interactions

During implantation, the embryonic trophoblast aggressively invades the uterine stroma. The resulting uterine reaction, decidualization, involves differentiation of new cell morphologies and remodeling of the extracellular matrix. This creates an environment that first permits invasion, then controls this invasion to allow the establishment of the placenta. The production, organization, and cellular interactions with the matrix are thought to underlie decidual functions. We have begun a reductional analysis of the components of the decidual matrix, focusing on extra‐cellular fibronectin (FN). Using decidual cell cultures prepared from day 7 implantation sites, the synthesis, extracellular organization, and details of decidual cell:FN interaction were studied. Employing immunofluorescence, immunoprecipitation, and dot blot analysis, decidualizing cultures showed a constitutive level of FN synthesis and deposition. The differentiating cells organized extracellular FN in patterns similar to that seen in vivo. The predominant, flattened dendritic decidual cells organized FN in long, thin fibrils. Large, rounded decidual cells, limited to the primary decidual zone in vivo, showed FN limited to punctate membrane patches and short, thick fibrils. Using double labeling techniques, FN expression was co‐localized with actin microfilament (MF) bundles during the cytoskeletal changes associated with the differentiation of both decidual cell types. The function of MFs in maintaining morphology was demonstrated by cytochalasin B perturbation. Attachment of decidual cells to FN was calcium dependent and gly‐arg‐gly‐asp‐ser‐pro (GRGDSP) sensitive, with dendritic decidual cells expressing the α5 and β1 integrin subunits. This suggests that an integrin system functions to attach decidual MF bundles to extracellular FN. This work shows that during decidual matrix remodeling, constitutive levels of FN are maintained to provide an extracellular framework to stabilize the decidual cytoskeleton and support morphological differentiation of decidual cells.

Pitfalls and improved techniques in avian parentage studies

-Recent studies on avian parentage have used both biochemical techniques and field observations to detect intraspecific brood parasitism and extra-pair fertilizations (EPFs). In long-term parentage studies of the European Starling (Sturnus vulgaris) using electrophoresis and field observations, we have encountered several methodological problems. Based on our findings and the existing literature, we describe these pitfalls and suggest improvements in techniques. The pitfalls include difficulty in obtaining large sample sizes, manipulation of natural populations, inaccurate allelic frequencies based on electrophoretic screening of too few individuals, catching the wrong putative parents, improper storage and handling of tissue samples, confounding effects of developmental changes in allelic expression, and misinterpretation of electrophoretic results. To improve parentage studies, we suggest censusing nest boxes around the peak times of laying to detect more than one egg per day, matching nestlings with eggs, establishing strict criteria for identifying putative parents at a nest, collecting pectoral muscle using an incision that goes in the direction of the superficial pectoral muscle fibers, and assessing the relative efficiency of laboratory and field methods to decide which would yield maximum results. Received 4 March 1988, accepted 6 September 1988. THE use of electrophoresis to study avian parentage has increased rapidly (Gowaty and Karlin 1984, Fleischer et al. 1985, Gavin and Bollinger 1985, Joste et al. 1985, Mumme et al. 1985, Evarts and Williams 1987, Westneat 1987, Wrege and Emlen 1987, Kendra et al. 1988). More recently, DNA technology has been applied to birds for the same purpose (Quinn et al. 1987, Wetton et al. 1987). The goal is to detect intraspecific brood parasitism and extra-pair fertilizations (EPFs). Intraspecific brood parasitism involves egg laying in the nest of a conspecific with no subsequent parental investment by the parasite (Yom-Tov 1980). EPFs can, but do not always have to, produce cuckoldry (Power et al. 1981). The detection of intraspecific brood parasitism or EPFs is an indication that several reproductive strategies exist in a population of birds. Comparing protein phenotypes and DNA fragment patterns between putative parent and offspring for the purpose of parental exclusion may be the only way to detect these phenomena in some cases. This is especially true of EPFs where observation (such as witnessed copulation) cannot confirm parentage. In the case of intraspecific brood parasitism, both biochemical techniques (e.g. electrophoresis and DNA fingerprinting) and field observations may be used to detect parasite eggs or offspring. The general methods of parentage studies are to collect eggs for studies of maternity only (Fleischer et al. 1985, Kendra et al. 1988) or to collect tissue (such as blood, pectoral muscle, feather pulp, or liver) from adults and nestlings for studies of maternity or paternity (Gowaty and Karlin 1984, Gavin and Bollinger 1985, Mumme et al. 1985, Evarts and Williams 1987, Quinn et al. 1987, Westneat 1987, Wetton et al. 1987, Wrege and Emlen 1987). The collected eggs or tissues are then examined for evidence of nonparentage. Field observations may be used as additional data or to supplement existing biochemical data (Quinn et al. 1987). Our purpose was to review the pitfalls of techniques used to study avian parentage and to suggest improved techniques based on our own experiences with European Starlings (Sturnus vulgaris). This review primarily pertains to field methods and electrophoresis. It is apparent from the recent literature that researchers may not be aware of all of these difficulties or the need for improved techniques. We will refer to these studies (including our own) in the spirit of constructive criticism. We feel strongly that 129 The Auk 106: 129-136. January 1989 This content downloaded from 157.55.39.243 on Thu, 06 Oct 2016 04:41:40 UTC All use subject to http://about.jstor.org/terms 130 ROMAGNANO, McGuIRE, AND POWER [Auk, Vol. 106 understanding the drawbacks of certain practices before they become common in parentage studies is important.

INTERACTION OF MOUSE ECTOPLACENTAL CONE TROPHOBLAST AND UTERINE DECIDUA IN VITRO

SummaryDuring the peri-implantation stages of mouse development, the secondary trophoblast invades into the uterine decidua. This uniquely controlled invasive process results in the formation of the placenta. We have analyzed this process in vitro using cultures of decidua and microdissected ectoplacental cones from Day 7 embryos. The results showed that the interaction between these two cell types is comparable to that seen in vivo. Morphologically, the decidua maintained close contact with the spreading trophoblast, limiting its invasion and producing a multilayered trophoblast outgrowth. Attachment to the decidua was not mediated through cell-matrix binding, but the subsequent invasion into the decidua was dependent on normal matrix interactions. Secretion of proteinases by the trophoblast also seemed to be a requirement for successful invasion, but not attachment.

Suspected infanticide in the starling

Avian adult infanticide (the killing of conspecific young by an adult) has been reported for several species (e.g., Crook and Shields 1985, Loftin and Roberson 1983, Trail et al. 1981). Here we document one case of attempted infanticide and circumstantial evidence for 3 more cases of infanticide in the European Starling (Sturnus vulgaris). These observations were made during an ongoing study of the behavioral ecology of the starling in Piscataway, New Jersey (Power et al. 1981), from 1983 to 1985. Nest boxes were attached to utility poles found along roads and mowed fields. As part of our studies on intraspecific brood parasitism, boxes were censused daily during laying. Adults were captured after all their eggs hatched and marked with numbered U.S. Fish and Wildlife Service bands and unique combinations of color bands. On 10 May 1984, we banded the breeding female at nest box 1-18. On 11 May 1984, we banded the breeding male and again saw the banded female. This pair had three, five-day-old nestlings. Minutes after the male was banded and released at 16:30, we observed an unbanded female fly to the nest box top (starlings can be sexed from a distance by polymorphism in bill color [Bullough 1942]). She entered the nest box, looked out ofthe entrance hole several times, and then exited to the ground with something in her mouth which she attempted, but failed to eat. (We later found this object to be a fragment of an unhatched egg with dried remains of an embryo.) She then reentered the box, exited quickly, and flew out of sight at 16:40. The breeding adults at this box were not present during this time. As soon as the unmarked female left, we examined the chicks and found that one nestling had a fresh bloody wound near the auditory opening but appeared otherwise healthy; the other two nestlings were unharmed. We examined the contents of the box during the capture of the adult male at 16:30 and observed no irregularities. Therefore, we conclude that the wound was inflicted by the visiting female between 16:30 and 16:40. On 18 May 1984, at box 1-20 a twelve-day-old nestling was discovered with a wound similar to that of the chick in box 1-18, about 80 m away. This wound had to have been inflicted one or more days earlier because the eye nearest to the damaged ear was swollen and infected. Unlike the first nestling described, this nestling died. On 8 June 1983, a dead chick about five days old was found on the ground between two nest boxes, I-13 and I14, with a bad wound over its left eye. This chick was too young to have fledged or even crawled out of its nest. Moreover, no chicks were missing from any of our boxes so this chick must have been carried some distance from its nest. On 5 June 1985, we found a dead four-day-old chick at box 111-12. The area below the chicks left auditory opening was covered with blood. This chicks nestmates had already died by 4 June of apparent starvation. Their mother was banded on 3 June. A male was never captured at this box. On 6 June, an unbanded female was captured in the box and fresh nesting material was present in the nest cup. This female began laying on 12 June. In all cases, the nature of the wound was the same: a peck mark near one of the auditory openings. Similar wounds have been reported by Crook and Shields (1985), Shelley (1934), and Stacey and Edwards (1983) in the context of infanticide. The possibility that mammals or different bird species may have caused the wounds in the three cases for which we have evidence for infanticide seems remote. The only other bird that has ever been observed to enter one of our starling boxes was an American Kestrel (Falco sparverius); it was successfully repelled by the adults at that box. To discourage mammalian predators such as Raccoons (Procyon lotor) and Gray Squirrels (Sciurus carolinensis), aluminum predator guards have been placed around utility poles below nest boxes. Squirrels still occasionally present a problem. However, they always remove the entire contents of the nest (either eggs or nestlings) and disrupt the nest itself. In addition, similar peck marks have been found around the head and neck of dead adults (n = 3) found in nest boxes, and on the faces of pairs of birds that we have captured fighting inside boxes (n = 8 pairs), suggesting that bill stabbing is not uncommon. In two of the four cases, a female was suspected to have committed an infanticidal act. We suspect that infanticide in our population is related to competition for nest sites, a limiting resource (e.g., see Hrdy 1979). Other reports of female infanticidal behavior have been reported by Loftin and Roberson (1983), and Picman (1977). In both cases, a limiting resource (such as nests, mates or food) has been suggested as a reason for infanticidal acts. The importance and frequency of avian infanticide is largely unknown primarily because the observation time necessary to document this phenomenon can be prohibitive. As a result, infanticide may have greater biological significance than is implied by the existing literature. We encourage all observers to be cognizant of the possibility of infanticide in their study populations.

Cellular Interactions and the Cysteine Proteinases in the Process of Mouse Implantation

In the mouse, implantation and placentation require the invasion of the uterine stroma by trophoblast giant cells. Concurrent with this invasion, the uterine stroma undergoes the process of decidualization. Through changes in cell morphology and matrix remodeling, this tissue forms a barrier to invasion and much of the maternal vasculature associated with the placenta. Both uterine remodeling and trophoblast invasion are proteinase dependent events, requiring the synthesis and secretion of a number of different enzymes. The work summarized here focuses on the expression, localization, function, and possible control of the cysteine proteinases, cathepsins B and L, during mouse implantation and placentation.