Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

Abstract

BackgroundIn Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation.ResultsA total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters.ConclusionsOur Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.

Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

“…The passive diffusion of potassium to the uterine lumen through the apical membrane is induced by a high concentration of intracellular potassium in cells (>100 mM). Five gene products in connection with the transfer of potassium, during its secretion into the uterine lumen, were detected in this study (Table 2, Figure 3) and confirmed previous observations of over-expression of two of these voltage-dependent ion channels regulated by the extracellular K + (KCNJ2, KCNJ16; Table 2 and Figure 3) and of the alpha subunit 1 of large conductance calcium activated potassium channel (KCNMA1) [23]. Their activation would modulate the export of potassium thus contributing to the membrane repolarization.…”

“…Indeed, potassium is secreted into the uterine fluid during the calcification of the shell and its concentration varies in this milieu from 12 mM at the initial phase of calcification and increases to 60 mM during the active growth phase [22]. Over-expression of the α1 and β1 subunits of the Na/K ATPase (ATP1A1 and B1) has been demonstrated in the uterus of laying hens during shell calcification [23,55] but was not statistically different in our study (N/E ratio 1.5). In contrast, we observed the over-expression (N/E ratio 1.92; Table 2), of NKAIN4, belonging to the NKAIN family.…”

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

“…Another group of proteins upregulated in SM‐Strong or UF‐Strong are about calcium binding and regulation. Calcium (Ca 2+ ), together with bicarbonate ion (HCO 3 − ), is the main component for the calcification of calcite layer of eggshell . Calcium‐binding property is usually a precondition for matrix proteins to participate in calcium biomineralization.…”

The pattern of distribution and interaction of metals and calcium in eggshells and egg contents in relation to the embryonic development of eggs in a small passerine bird

“…Calcium sequestration in the biochemical/physiological context is attributable to the same ion transporter(s) that bind(s) a few different elements that are simultaneously deposited in the eggshell (cf. Scheuhammer 1996; Rodriguez-Navarro et al Jonchere et al 2012). During oogenesis in female birds, the absorption of certain non-essential Ca-mimetic metals (like Pb or Cd) increases during Ca sequestration into shells (Scheuhammer 1996).…”