Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Sex- and tissue-specific expression of "similar to nothepsin" and cathepsin D in relation to egg yolk formation in Gallus gallus.

Egg yolk constitutes the main storage compartment of the avian egg and the first nutritional source that supports embryonic growth. Most egg yolk components are synthesized by the liver of laying hens at sexual maturity and are secreted into the blood to be further transferred into the ovarian oocyte (yolky follicle) by receptor-mediated endocytosis. Egg yolk proteins are secreted as precursors and must undergo proteolytic processing to be bioactive. It is assumed that chicken cathepsin D, an aspartic protease, is a key enzyme in this process. Very recently, a novel aspartic protease, namely "similar to nothepsin," has been identified in the egg yolk. Previous experiments conducted in Antarctic fish have shown that the expression of nothepsin is tissue- and sex-specific. To gain insight into the specificities of expression of both cathepsin D and "similar to nothepsin" in Gallus gallus, we compared their distribution in various tissues, in male and females. Cathepsin D is ubiquitously expressed in all tissues examined, including liver of both male and female adults, and its expression is stable during sexual maturation. In contrast, "similar to nothepsin" expression is unique to the liver of adult females and is sex steroid-dependent as it increases gradually in the liver of hens during sexual maturation. The sexual dimorphic expression of the "similar to nothepsin" gene suggests that the activity of this protein is regulated by the steroid environment of laying hens and is specifically adapted for inclusion in the yolk. Further studies are needed to assess whether "similar to nothepsin" assists cathepsin D in the proteolytic processing of egg yolk proteins during follicular growth.

Review: Production factors affecting the quality of chicken table eggs and egg products in Europe.

The hen's egg (Gallus gallus) is an animal product of great agronomic interest, with a world production of 70.9 million tonnes in 2018. China accounted for 35% of world production, followed by North America (12% of world production), the European Union (7.0 million tonnes, 10% of world production) and India (5.0 million tonnes, 7% of world production). In France, 16-17 billion eggs are produced annually (14.5 billion for table eggs) and more than 1 200 billion worldwide. In 2019, egg production increased by 3.3% compared to 2018, mainly due to the increase in Asian production, which has risen by 42% since 2000. Chicken eggs are widely used either as a low-cost, high nutritional quality food cooked by the consumer (more than 100 billion eggs consumed in Europe), or incorporated as an ingredient in many food products. The various production methods have changed considerably over the last 15 years with the consideration of animal welfare and changes in European regulations. In Europe, fewer and fewer eggs are produced in confinement and there has been a strong growth in the number of systems giving access to an outdoor run. In this review, we describe the different ways in which eggs are produced and processed into egg products to meet the growing demand for ready-to-use food products. We analyse the effect of this evolution of hen-rearing systems on the set of characteristics of eggs and egg products that determine their quality. We describe the risks and benefits associated with these new production methods and their influence or lack of influence on commercial, nutritional, microbial and chemical contamination risk characteristics, as well as the evolution of the image for the consumer. The latter covers the ethical, cultural and environmental dimensions associated with the way the egg is produced.

Review: Quality and authentication of organic animal products in Europe.

The 'organic' label guarantees a production process that avoids the use of synthetic fertilisers, pesticides and hormones and minimises the use of veterinary drugs; however, consumers are demanding guarantees regarding food quality. This article reviews the current state of knowledge on the quality of organic animal products, including the authentication of their organic origin. Quality has been considered as an integrative combination of six core attributes: commercial value, and nutritional, sensory, technological, convenience and safety attributes. The comparison of these attributes between organic and conventional animal products shows high heterogeneity due to variability in farming pratices in both organic and conventional systems. To overcome this, we pinpoint the farming practices underlying the differences observed. This enables light to be shed on the consequences of possible trajectories of organic farming, if specifications are relaxed or tightened up on commitments concerning farming practices that impact product quality. Two recent meta-analyses showed better nutritional attributes in organic milk and meat linked to their higher poly-unsaturated fatty acid (PUFA) content, particularly n-3 PUFAs. Regarding safety, we point to a lack of integrated studies quantifying the balance between positive and negative effects. Organic farming reduces the risk of drug residues and antibiotic resistance, but both outdoor rearing and a frequently longer rearing period increase the animals' exposition to environmental contaminants and the risk of their bioaccumulation in milk, eggs, meat and fish flesh. We highlight antagonisms between quality attributes for certain animal products (lamb, pork). In general, attributes are more variable for organic products, which can be explained by lower genetic selection (poultry), lower inputs and/or greater variability in farming conditions. However, the literature does not address the implications of this greater variability for the consumers' acceptability and the necessary adaptation of manufacturing processes. Further research is needed to document the impacts on human nutritional biomarkers and health. Methods used to authenticate organic origin are based on differences in animal diet composition between organic and conventional systems, but their reliability is hampered by the variability in farming practices.

Review: Quality of animal-source foods.

This article critically reviews the current state of knowledge on the quality of animal-source foods according to animal production and food processing conditions, including consumer expectations-behaviours and the effects of consumption of animal-source foods on human health. Quality has been defined through seven core attributes: safety, commercial, sensory, nutritional, technological, convenience, and image. Image covers ethical, cultural and environmental dimensions associated with the origin of the food and the way it is produced and processed. This framework enabled to highlight the priorities given to the different quality attributes. It also helped to identify potential antagonisms and synergies among quality attributes, between production and processing stages, and among stakeholders. Primacy is essentially given to commercial quality attributes, especially for standard commodity animal-source foods. This primacy has strongly influenced genetic selection and farming practices in all livestock commodity chains and enabled substantial quantitative gains, although at the expense of other quality traits. Focal issues are the destructuration of chicken muscle that compromises sensory, nutritional and image quality attributes, and the fate of males in the egg and dairy sectors, which have heavily specialised their animals. Quality can be gained but can also be lost throughout the farm-to-fork continuum. Our review highlights critical factors and periods throughout animal production and food processing routes, such as on-farm practices, notably animal feeding, preslaughter and slaughter phases, food processing techniques, and food formulation. It also reveals on-farm and processing factors that create antagonisms among quality attributes, such as the castration of male pigs, the substitution of marine-source feed by plant-based feed in fish, and the use of sodium nitrite in meat processing. These antagonisms require scientific data to identify trade-offs among quality attributes and/or solutions to help overcome these tensions. However, there are also food products that value synergies between quality attributes and between production and processing phases, particularly Geographical Indications, such as for cheese and dry-cured ham. Human epidemiological studies have found associations between consumption of animal-source foods and increased or decreased risk for chronic non-communicable diseases. These associations have informed public health recommendations. However, they have not yet considered animal production and food processing conditions. A concerted and collaborative effort is needed from scientists working in animal science, food process engineering, consumer science, human nutrition and epidemiology in order to address this research gap. Avenues for research and main options for policy action are discussed.

Review: What are the challenges facing the table egg industry in the next decades and what can be done to address them?

There has been a strong consumer demand to take welfare into account in animal production, including table eggs. This is particularly true in Europe and North America but increasingly around the world. We review the main demands that are facing the egg industry driven by economic, societal and sustainability goals. We describe solutions already delivered by research and those that will be needed for the future. Already table egg consumption patterns have seen a major shift from cage to non-cage production systems because of societal pressures. These often feature free-range and organic production. These changes likely signal the future direction for the layer sector with the acceleration of the conversion of cage to barn and aviary systems with outdoor access. This can come with unintended consequences from bone fracture to increased disease exposure, all requiring solutions. In the near future, the laying period of hens will be routinely extended to improve the economics and environmental footprint of production. Many flocks already produce close to 500 eggs per hens in a lifetime, reducing the number of replacement layers and improving the economics and sustainability. It will be a challenge for scientists to optimize the genetics and the production systems to maintain the health of these hens. A major ethical issue for the egg industry is the culling of male day-old chicks of layer breeds as the meat of the males cannot be easily marketed. Much research has and will be devoted to alternatives. Another solution is elimination of male embryos prior to hatching by in ovo sexing approaches. The race to find a sustainable solution to early stage sex determination is on. Methods based on sex chromosomes, sexually dimorphic compounds and spectral properties of eggs containing male or female embryos, are being researched and are reviewed in this article. Other proposed solutions include the use of dual-purpose strains, where the males are bred to produce meat and the females to produce eggs. The dual-purpose strains are less efficient and do not compete economically in the meat or egg market; however, as consumer awareness increases viable markets are emerging. These priorities are the response to economic, environmental, ethical and consumer pressures that are already having a strong impact on the egg industry. They will continue to evolve in the next decade and if supported by a strong research and development effort, a more efficient and ethical egg-laying industry should emerge.

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit.

BACKGROUND: The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents during eggshell biomineralization. Main Body. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. CONCLUSION: With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

Possible roles of parathyroid hormone, 1.25(OH)2D3, and fibroblast growth factor 23 on genes controlling calcium metabolism across different tissues of the laying hen.

This study provides an integrative description of candidate gene expression across tissues involved in calcium (Ca) metabolism during the egg laying cycle, using the well-defined model of Ca supply as fine or coarse particles of calcium carbonate (CaCO3). Plasma and tissue samples were collected from hens at the peak of laying at 0 to 1, 9 to 10, and 18 to 19 h postovulation (PO). After mRNA preparation from the parathyroid gland, medullary bone, liver, kidney, duodenum, and jejunum, gene expressions were quantified using RT-qPCR. The highest levels of parathyroid hormone (PTH) mRNA in the parathyroid gland (P < 0.05), and of the active form of vitamin D3 1.25(OH)2D3 in the plasma (P < 0.01) were observed at 18 to 19 h PO. During this active phase of eggshell formation, bone resorption was attested to high levels of plasma inorganic phosphorus (iP) and the receptor activation of nuclear factor-κB expression in the bone (P < 0.001 and P < 0.05, respectively). At this stage, 5 genes of the transcellular and the paracellular Ca absorption pathways in the intestine (P < 0.05) and the Ca channel transient receptor potential cation channel subfamily V member 5 (P < 0.05), involved in its reabsorption in the kidney, were overexpressed. At 0 to 1 h PO during the subsequent daylight period, 2 candidates of the transcellular and the paracellular Ca pathways (P < 0.05) remained at high levels in the intestine, while calbindin D 28K expression was the highest in the kidney (P < 0.05). As PTH mRNA and 1.25(OH)2D3 were low, bone accretion was likely active at this stage. The phosphaturic hormone fibroblast growth factor 23 (FGF23) was overexpressed at 18 to 19 h PO (P < 0.05) in the bone when plasma iP was high, which suggested a role in the subsequent reduction of P reabsorption in the kidney, as attested to the decreased expression of P cotransporters, leading to iP clearance from the plasma at 0 to 1 h PO (P < 0.05). The low levels of 1.25(OH)2D3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO3, higher plasma levels of 1,25(OH)2D3 and higher expression of several genes involved in bone turnover reflected a stronger challenge to Ca homeostasis. Altogether, these data support the hypothesis that FGF23 could drive vitamin D metabolism in the laying hen, as previously documented in other species and explain the tight link between P and Ca metabolisms.

Increased expression of fibroblast growth factor 23 is the signature of a deteriorated Ca/P balance in ageing laying hens.

The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.

Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens.

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116, osteopontin (SPP1), ovocalyxin-32 (RARRES1), ovotransferrin (LTF), ovalbumin and ovocalyxin-36, as well as key genes in the maintenance and function of the shell gland [estrogen receptor (ESR1) and carbonic anhydrase II (CAII)]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.

Candidate genes of the transcellular and paracellular calcium absorption pathways in the small intestine of laying hens.

To meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.

Growth factors in skeletal muscle regeneration.

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

Characterization of myogenesis from adult satellite cells cultured in vitro.

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.

Slow and fast rat skeletal muscles differ in their plasminogen activator activities.

Slow and fast contracting muscles differ in their innervation and electrophysiological properties as well as in their regenerating potentialities. The purpose of the present work was to investigate the expression of plasminogen activators and its possible relation to each type of muscle. Slow (Soleus) and fast (Extensor Digitorum Longus) muscles were obtained from white Wistar rats. Before sectioning the muscles, the euthanized rats were perfused with cold phosphate buffer saline to avoid interference by circulating proteases and inhibitors. Muscle extracts were pounded in an ice-cold Potter tube. Plasminogen activators (PAs) were assayed by fibrin zymography and by both liquid and solid-phase fibrin spectrophotometric assays for the detection of PAs activity. Both urokinase (uPA) and tissue-type plasminogen activator (tPA) activities corresponding to proteins of 38 kDa and 65 kDa molecular masses, were detected in the extracts. Slow muscles contained higher amounts of both activators than fast muscles, but the relative amount of uPA was higher in both types of muscles. In addition, the characteristics of each type of extracts differed somewhat: the fast muscle activity curve was typical of an accelerating process, while the slow muscle curve showed an activity probably related to already formed plasmin or to some other trypsin-like enzyme. These results suggest that the amount of plasminogen activators could be a new criterion of discrimination between slow and fast skeletal muscles.

Differential changes in protein kinase C associated with regeneration of rat extensor digitorum longus and soleus muscles.

We used a model of crush-induced regeneration in rat in order to characterize biochemically and histologically the implication of protein kinase C (PKC) in muscle repair after damage. In this model, slow soleus and fast extensor digitorum longus (EDL) muscle regeneration proceed differently. PKC activity has been assayed in regenerating muscles and their intact contralateral during the first 14 days following crushing. Degeneration (myolysis) occurring shortly after crush was associated with a marked down-regulation of the enzyme in both wound muscles and notable increase in the corresponding contralateral muscles. Muscle fiber reconstruction in EDL was associated with a rise in PKC activity which peaked at day 7 in regenerating muscle where it was twice higher than in intact muscle. At variance, muscle PKC activity in soleus increased slower than that of EDL and reached later intact level. Western blot analysis and immunohistochemical studies of representative members of the three PKC subfamilies were performed. All the isoform tested were much less expressed in regenerating than in control intact muscles suggesting that the overall PKC activity in regenerating muscles was more activable than in controls. We have shown that PKC isoforms were sequentially expressed during regeneration in both muscle types. PKC theta; being present the earliest, then delta, epsilon and alpha and finally zeta, beta and eta. Some isoforms were differentially expressed according muscle type. PKC delta being more expressed in soleus whereas beta and eta appeared earlier in EDL. Histochemical studies have revealed that the isoforms were differently localized in muscle tissue and that fiber regeneration was associated with PKC alpha translocation from sarcoplasma to sarcolemma. Together these data have shown that multiple PKC isoforms are implicated in the regenerative process acting at different in times and location and suggesting that individual isoform may fulfill distinct functions.

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Nature and bioactivity of gonadotropin-releasing hormone (GnRH) secreted during the GnRH surge.

Previous studies have demonstrated a neural action of estradiol in inducing a surge of GnRH in the ewe. However, although the GnRH and LH surges began concurrently, the GnRH surge consistently continued well beyond the surge of LH. Three experiments were conducted to test the hypothesis that the termination of the LH surge results from the secretion of a relatively inactive variant of GnRH during the later phases of the GnRH surge. In the first experiment, hypophyseal portal blood collected during an estrogen-induced LH surge was analyzed for GnRH immunoreactivity using two antibodies having specificity for the N- or C-terminal portion of the GnRH molecule. The duration, amplitude, and time course of the GnRH surge were found to be similar irrespective of the antisera used. In a second experiment, a competitive GnRH antagonist was administered at the beginning of the estrogen-induced GnRH/LH surge at a dose capable of blocking pituitary responsiveness for approximately half the duration of the GnRH surge. Antagonist treatment did not result in any change in the time of onset of the GnRH surge, but there was no increase in LH that naturally occurs coincident with onset of the GnRH surge. Rather, a persistent increase in LH secretion was observed during the latter stages of the GnRH surge, indicating that the GnRH molecules secreted at this time were biologically active. Finally, a sensitive and specific ovine pituitary cell bioassay was used to test bioactivity of GnRH in hypophyseal portal blood during different phases of the GnRH surge. GnRH bioactivity in samples collected early in the GnRH surge was greater than that before the onset of the GnRH surge but no greater than that collected during the descending limb of the surge. The results of all three experiments fail to support the hypothesis that the LH surge ends because of a change in the nature of the GnRH secreted. Rather they show that GnRH secreted throughout the surge is biologically active. Thus, the termination of the LH surge before that of the GnRH surge occurs for reasons other than lack of a bioactive GnRH signal.

Normal and growth hormone (GH)-secreting adenomatous human pituitaries release somatostatin and GH-releasing hormone.

Neuropeptides such as vasoactive intestinal peptide, LHRH, or TRH have been found in rat pituitary tissue and could act via paracrine or autocrine actions in this tissue. In this study we investigated whether normal human pituitary tissue and GH-secreting human pituitary adenomas could release somatostatin (SRIH) and GHRH. Fragments from three human pituitaries and dispersed cells from six GH-secreting adenomas (four adenomas were studied for GHRH release and five for SRIH release) were perifused using a Krebs-Ringer culture medium, and the perifusion medium was collected every 2 min (1 mL/fraction for 5 h). GH, GHRH, and SRIH were measured by RIA under basal conditions and in the presence of 10(-6) mol/L TRH or SRIH. Both normal pituitaries and GH-secreting pituitary adenomas released SRIH and GHRH. SRIH release commenced 90-180 min after initiation of the perifusion, at which time GH secretion had decreased significantly. TRH stimulated SRIH release from normal pituitary tissue and inhibited SRIH release from adenoma tissue. GHRH was present at the start of the perifusion, but rapidly disappeared. However, SRIH stimulated GHRH release from normal pituitary tissue, but not from adenoma tissue. Significant amounts of GHRH and SRIH were released during the experiments, suggesting their local synthesis. These results indicate that pituitary cells can release hypothalamic peptides. The liberation of these neuropeptides is regulated, and moreover, their regulation differs between normal and adenomatous pituitaries.

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.