The placental analogue and the pattern of sexual reproduction in the cheilostome bryozoan Bicellariella ciliata (Gymnolaemata)

Background

Matrotrophy or extraembryonic nutrition ?C transfer of nutrients from mother to embryo during gestation ?C is well known and thoroughly studied among vertebrates, but still poorly understood in invertebrates. The current paper focuses on the anatomy and ultrastructure of the oogenesis and placentotrophy as well as formation of the brood chamber (ovicell) in the cheilostome bryozoan Bicellariella ciliata (Linnaeus, 1758). Our research aimed to combine these aspects of the sexual reproduction into an integral picture, highlighting the role of the primitive placenta-like system in the evolution of bryozoan reproductive patterns.

Results

Follicular and nutrimentary provisioning of the oocyte occur during oogenesis. Small macrolecithal oocytes are produced, and embryos are nourished in the ovicell via a simple placental analogue (embryophore). Every brooding episode is accompanied by the hypertrophy of the embryophore, which collapses after larval release. Nutrients are released and uptaken by exocytosis (embryophore) and endocytosis (embryo). Embryos lack specialized area for nutrient uptake, which occurs through the whole epidermal surface. The volume increase between the ripe oocyte and the larva is ca. 10-fold.

Conclusions

The ovicell is a complex organ (not a special polymorph as often thought) consisting of an ooecium (protective capsule) and an ooecial vesicle (plugging the entrance to the brooding cavity) that develop from the distal and the fertile zooid correspondingly. Combination of macrolecithal oogenesis and extraembryonic nutrition allows attributing B. ciliata to species with reproductive pattern IV. However, since its oocytes are small, this species represents a previously undescribed variant of this pattern, which appears to represent a transitional state from the insipient matrotrophy (with large macrolecithal eggs) to substantial one (with small microlecithal ones). Altogether, our results substantially added and corrected the data obtained by the previous authors, providing a new insight in our understanding of the evolution of matrotrophy in invertebrates.     

Prudent female allocation by modular hermaphrodites: female investment is promoted by the opportunity to outcross in cyclostome bryozoans

Many sessile, suspension‐feeding marine invertebrates mate by spermcasting: aquatic sperm are spawned and gathered by conspecific individuals to fertilize eggs that are generally retained during development. In two phylogenetically distant examples, a cheilostome bryozoan and an aplousobranch ascidian, the receipt of allosperm has previously been shown to alter sex allocation by triggering female investment in eggs and brooding. Here we report experiments demonstrating that two species of cyclostome bryozoan also show restrained female investment in the absence of mating opportunity. In Tubulipora plumosa, the production of female zooids and progeny is much reduced in reproductive isolation. In Filicrisia geniculata, development of distinctive female zooids (gonozooids) begins but halts in the absence of mating opportunity, and no completed gonozooids or progeny result. Reduced female investment in the absence of a mate thus occurs in at least two orders of Bryozoa, but significant differences in detail exist and the evolutionary history within the phylum of the mechanism(s) by which female investment is initiated might be complex. The broadening taxonomic spectrum of examples where female investment appears restrained until allosperm becomes available may signify a general adaptive strategy among outcrossing modular animals, analogous to similarly adaptive sex allocation typical of many flowering plants.     

Comparative Studies of Ovicell Anatomy and Reproductive Patterns in Cribrilina annulata and Celleporella hyalina (Bryozoa: Cheilostomatida)

Investigations of the common boreal-arctic cheilostomate bryozoans Cribrilina annulata and Celleporella hyalina have shown that the two species possess similar ovicell structures and reproductive patterns. Both species are characterized by frontal dwarf ovicellate zooids, that are female autozooidal polymorphs in C. hyalina and simultaneous hermaphroditic autozooids in C. annulata. The latter species in addition has ovicellate autozooids of the usual type. Each ovicell is formed from a maternal zooid only, and its cavity is lined by the outer hemispherical fold (ooecium) and the distal zooidal wall. The coelomic cavity of the ooecium is separated from the body cavity of the maternal zooid by a transverse wall with simple pores. Each pore is closed by a cell plug, and the ooecia may be considered as kenozooids. Each oocyte is accompanied by a single nurse cell that degenerates after ovulation. The eggs are macrolecithal in C. annulata and microlecithal in C. hyalina, and the former species is a non-placental brooder whereas the latter forms a placenta. Fertilization is precocious. Possible mechanisms of sperm entry as well as oviposition are discussed. The literature concerning ovicell structure and development in cheilostomates is analysed. It is proposed that the brood chamber of cribrimorphs evolved by a fusion of costae and a reduction of the daughter zooid in ancestral forms. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Oogenesis in a placental viviparous onychophoran

This first ultrastruetural study of oogenesis in a placental viviparous onychophoran describes oocyte differentiation, cell interactions and reveals various unusual cellular features. The viviparous onychophoran Plicatoperipatus jamaicensis has paired ovaries medially located, attached to the dorsal body wall by muscular terminal filaments. The rest of the female reproductive tract consists of paired spermathecae oviduct/uteri (hereafter referred to as uterus). Bulbous spermathecae are joined to the oviducts by ducts. Also continuous with the oviduct lumen are two tubular structures whose lumina open to the hemolymph. The uteri contain a progression of developmental stages from implantation through stalked morulae, blastocysts, larvae and juveniles about to be born.Growing oocytes are characterized by large germinal vesicles showing synaptonemal complexes. Oocytes are surrounded by flattened follicle cells that possess extensive bundles of thick and thin filaments. Mature oocytes contain little or no yolk, but are unique among organisms in accumulating a large central reservoir of stored glycogen. The lack of yolk reflects the placental viviparous nature of the reproductive process. The glycogen reservoir provides a rapidly accessible energy source for early developmental stages.Particularly prominent also are unusually extensive and highly elaborate Golgi complexes in the cortical and peri-nuclear ooplasm. While extensive Golgi complexes have been described in oocytes of a variety of species, the particularly exaggerated size and amount of Golgi in these onychophorans suggests they may provide excellent material for the study of Golgi function. The features of the oocyte and placental viviparity show this is an ideal model to investigate the nature of the placental reproductive process analogous to mammals in an invertebrate and its implications to oogenesis.     

Fitness consequences of allorecognition-mediated agonistic interactions in the colonial hydroid Hydractinia [GM

In sessile and sedentary organisms, competition for space may have fitness consequences that depend strongly on ecological context. Colonial hydroids in the genus Hydractinia use an inducible defense when encountering conspecifics, and intraspecific competition is common in natural populations, often resulting in complete overgrowth of subordinate competitors. My goal in this study was to quantify the impacts of agonistic interactions in Hydractinia [GM] (an undescribed species from the Gulf of Mexico) in terms of three primary fitness components: colony survival, growth rate, and immature gonozooid production. The results demonstrate that the fitness consequences of intraspecific competition depend on the size at which competitive encounters are initiated and the growth form (an indicator of competitive ability) of the competitors. Moreover, some competing colonies consistently produced more immature gonozooids than the controls without competition, and they exhibited extremely low mortality even after 90 days of growth. These results have several ramifications. First, agonistic interactions do not always proceed to competitive elimination. Second, the increase in production of immature gonozooids--an investment in future reproduction--in response to intraspecific competition supports the hypothesis that indeterminately growing organisms increase sexual reproductive effort when growth becomes limiting. Lastly, in light of known ontogenetic variation in the ability of Hydractinia to differentiate among genetically related colonies, strongly size-dependent fitness consequences are consistent with an adaptive, kin-discriminating allorecognition system.     

Prp-c and Prp-Sc at the fetal-maternal interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.     

Jet propulsion in Doliolum (Tunicata: Thaliacea)

The jet propulsion of doliolid gonozooids is described from a combination of kinematic, chamber pressure, and muscle electrical records. Doliolids respond to light mechanical stimuli by a rapid contraction of the locomotor muscle bands, producing a single jet pulse which drives the animal forwards or backwards at instantaneous velocities up to 21.4cm·s^?1 (over 50 body lengths·s^?1). Spike potentials from the (multiply innervated) locomotor muscle fibres are variable in size and probably are non-propagating. Maximum chamber pressures during the jet pulse range up to 500 Pa, doliolids ≈4.5 mm long perform around 4 × 10^?6 J work per contraction. Although the locomotor system is specialized for single rapid escape movements, the same movements are used at irregular intervals to maintain the horizontal position of the animal (which is denser than sea water) in the water column. The locomotor system is less economical than that of salps.     

GM-CSF regulation of embryo development and pregnancy

The reproductive tissues undergo profound structural changes and major immune adaptation to accommodate pregnancy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of an array of cytokines with pivotal roles in embryo implantation and subsequent development. Several cell lineages in the reproductive tract and gestational tissues synthesise GM-CSF under direction by ovarian steroid hormones and signalling agents originating in male seminal fluid and the conceptus. The pre-implantation embryo, invading placental trophoblast cells and the abundant populations of leukocytes controlling maternal immune tolerance are all subject to GM-CSF regulation. GM-CSF deficiency in pregnancy adversely impacts fetal and placental development, as well as progeny viability and growth after birth, highlighting this cytokine as a central maternal determinant of pregnancy outcome with clinical relevance in human fertility.     

Ultrastructure and Development of the Ooecial Walls in Some Calloporid Bryozoans (Gymnolaemata: Cheilostomata)

In the brood chambers (ovicells) of six calloporid cheilostomes studied each skeletal wall consists of four calcified layers: (1) a very thin superficial layer of planar spherulitic crystallites, (2) an upper (outer) layer with wall-perpendicular prismatic ultrastructure, (3) an intermediate lamellar layer, and (4) a lower (inner) wall-perpendicular prismatic layer. Comparative studies of both the ovicell wall ultrastructure and early ovicell formation showed a hypothetical opportunity for evolving complex (multilayered) skeletal walls by fusion of the initially separated gymnocystal and cryptocystal calcifications in Cheilostomata. In two species studied, a bilobate pattern in the final stage of the formation of the ooecial roof was encountered in specimens with the cuticle preserved. A possible explanation to this finding is discussed – the bilobate pattern is suggestive of the hypothetical origin of the brood chamber from (1) two flattened spines, or (2) reduction in spine number of an originally multispinous ovicell.     

Study of the mechanism of postcoital contraception in the combination of streoids and the central m-cholinolytic, amizil]

Experiments on female rats showed the blocking of the M-cholinoreactive system with amizyl to significantly contribute to the estrogen/norsteroid contraceptive effect during the postcoital periods. This effect was accompanied by decrease in the gonadotropin level and by the change in the LH/FSH ratio, this creating an unfavourable background for implantation of the fertilized ovicell in the endometrium. There was a change in the transport rate in the tube and a delay in the decidual reaction. Changes in the rate of the ovicell transport were not accompanied by distrubances in the process of fertilization or with the cytotoxic action. Mestranol and ethynylestradiol in combination with norethynodrel (1:20) and with the central cholinolytic amizyl were agents with future prospects for short-term postcoital contraception.     

Rural populations of the red fox Vulpes vulpes show little evidence of reproductive senescence

The ageing theory predicts fast and early senescence for fast-living species. We investigated whether the pattern of senescence of a medium-sized, fast-living and heavily-culled mammal, the red fox (Vulpes vulpes), fits this theoretical prediction. We used cross-sectional data from a large-scale culling experiment of red fox conducted over six years in five study sites located in two regions of France to explore the age-related variation in reproductive output. We used both placental scars and embryos counts from 755 vixens’ carcasses aged by the tooth cementum method (age range: 1–10), as proxies for litter size. Mean litter size per vixen was 4.7 ± 1.4. Results from Generalized Additive Mixed Models revealed a significant variation of litter size with age. Litter size peaked at age 4 with 5.0 ± 0.2 placental scars and decreased thereafter by 0.5 cubs per year. Interestingly, we found a different age-specific variation when counting embryos which reached a plateau at age 5–6 (5.5 ± 0.2) and decreased slower than placental scars across older ages, pointing out embryo resorption as a potential physiological mechanism of reproductive senescence in the red fox. Contrary to our expectation, reproductive senescence is weak, occurs late in life and takes place at an age reached by less than 11.7% of the population such that very few females exhibit senescence in these heavily culled populations.     

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.     

Toll-like receptors in the gonads and reproductive tract: emerging roles in reproductive physiology and pathology

Interactions between the immune system and reproductive system have important consequences for fertility and reproductive health in general. There is increasing evidence that many of the interactions between the immune and reproductive systems involve the Toll-like receptors (TLRs). While there is no doubt that TLRs are important in providing protection against infection in the reproductive tract, there is increasing evidence for the involvement of TLRs in more basic pathology and physiology of reproduction. In the female, TLRs have been implicated in critical aspects of ovarian, endometrial and placental function, as well as in ovarian cancer, pelvic inflammatory disease, intrauterine growth restriction, pre-eclampsia and preterm birth. In the male, TLRs appear to play a role in the control of testicular steroidogenesis and spermatogenesis in disease and, potentially, during normal function, as well. Recent studies also have begun to highlight the role of various TLRs in the aetiology of prostatitis and prostatic cancer. Given the nascent state of knowledge concerning this important area, it is clear that more studies are needed, which should provide valuable new insights into the biology of the TLRs and reproductive function in general.     

Pregnancy initiation in the rhesus macaque: Towards functional manipulation of the maternal-fetal interface

Nonhuman primates provide an important opportunity to define the mechanisms that contribute to the success of early pregnancy. We have focused for several years now on defining the expression of novel placental major histocompatibility complex (MHC) class I molecules. In parallel, we have used reagents against human immune cell markers to characterize the leukocyte population in the decidua and have demonstrated dynamic changes in these cell populations during the first 5 weeks of gestation. The challenge is to identify the possible role(s) of placental MHC class I in modifying/directing the maternal endometrial or systemic immune system in the post-implantation period. Foremost among the challenges is the difficulty in modifying placental function. In the instance of trophoblast surface proteins, passive immunization studies are feasible, although limitations include the empirical nature of this approach, as well as the inability to modify intracellular function. We have shown that using lentiviral vectors to effect preimplantation gene transfer for transgene expression in the placenta is not only feasible, but of good efficiency. In addition to transgene overexpression, robust approaches for knocking down/knocking out placental gene expression are essential. Recent developments in RNA interference approaches may allow "transient knockout" experiments. While the rhesus monkey has been our model of choice, currently there are limitations in the number of available female rhesus monkeys of reproductive age for research in early pregnancy. It is critical that the technologies for advanced study move forward in other species. The baboon has been used significantly in reproductive tract biology and early pregnancy research and important models have been developed for manipulation of the maternal-fetal interface. Additional characterization of other species, such as the cynomolgus and African green (vervet) monkey is critical. Given the limitations on antigen recognition when using human reagents, we also propose that the development of panels of primate-specific anti-leukocyte antibodies is essential for moving forward nonhuman primate reproductive research.     

Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.     

The effect of leptin on mouse trophoblast cell invasion

The hormone leptin is produced by adipose tissue and can function as a signal of nutritional status to the reproductive system. The expression of leptin receptor and, in some species, leptin, in the placenta suggests a role for leptin in placental development, but this role has not been elucidated. Leptin is required at the time of embryo implantation in the leptin-deficient ob/ ob mouse and has been shown to upregulate expression of matrix metalloproteinases (MMPs), enzymes involved in trophoblast invasion, in cultured human trophoblast cells. This led us to the hypothesis that leptin promotes the invasiveness of trophoblast cells crucial to placental development. We found that leptin stimulated mouse trophoblast cell invasion through a matrigel-coated insert on Day 10, but not Day 18 of pregnancy. Optimal stimulation occurred at a concentration of 50 ng/ml leptin, similar to the peak plasma leptin concentration during pregnancy in the mouse. Leptin treatment did not stimulate proliferation of mouse trophoblast cells in primary culture. Leptin stimulation of invasion was prevented by 25 muM GM6001, an inhibitor of MMP activity. Our results suggest that leptin may play a role in the establishment of the placenta during early pregnancy and that this function is dependent on MMP activity. This effect of leptin may represent one mechanism by which body condition affects placental development.     

The genus Doliolina (Thaliacea: Doliolida)

The anatomical structure of the gonozooids of the differentspecies of the genus Doliolina has been re-examined. A divisionof the genus into two subgenera, Doliolina and Doliolinetta,based on the compared shape and position of the gonads, is proposedand a revised dichotomous key of the species is presented.