RESEARCH ARTICLE
Placental Lipoprotein Lipase (LPL) Gene Expression in a Placentotrophic Lizard, Pseudemoia entrecasteauxii OLIVER W. GRIFFITH1*, BEATA UJVARI2, KATHERINE BELOV2, 1 AND MICHAEL B. THOMPSON 1
School of Biological Sciences, University of Sydney, Sydney, NSW, Australia Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia
2
ABSTRACT
J. Exp. Zool. (Mol. Dev. Evol.) 320B:465–470, 2013
Viviparity (live birth) relies on a functional placenta, which is formed by cooperating maternal and embryonic tissues. In some viviparous lineages, mothers use this placenta to transport nutrients to feed developing embryos through pregnancy (placentotrophy). The Australian lizard, Pseudemoia entrecasteauxii, provides approximately 60% of the lipid for embryonic growth and metabolism to embryos across the placenta. Lipoprotein lipase (LPL) is an important enzyme in lipid transport in vertebrates. We examined patterns of LPL gene expression to identify its role in the uterus of pregnant P. entrecasteauxii. We used reverse transcription quantitative real time PCR to measure the expression of the LPL gene in the uterine tissue throughout reproduction and compared uterine LPL expression in chorioallantoic and yolk‐sac placentae. Expression of the LPL gene is significantly higher in the uterus of late pregnant compared to non‐pregnant and early pregnant P. entrecasteauxii, indicating a greater capacity for lipid transport towards the end of pregnancy. The period of high LPL gene expression correlates with the time that developing embryos are undergoing the greatest growth and have the highest metabolic rate. LPL gene expression is significantly higher in the uterine tissue of the yolk‐sac placenta than the chorioallantoic placenta, providing the first molecular evidence that the yolk‐sac placenta is the major site of lipid transport in pregnant P. entrecasteauxii. J. Exp. Zool. (Mol. Dev. Evol.) 320B: 465–470, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Griffith OW, Ujvari B, Belov K, Thompson MB. 2013. Placental lipoprotein lipase (LPL) gene expression in a placentotrophic lizard, Pseudemoia entrecasteauxii. J. Exp. Zool. (Mol. Dev. Evol.) 320B:465–470.
Oviparity (egg laying) is the primitive reproductive state for amniote vertebrates (reptiles, birds, and mammals). Viviparity (live birth) has evolved independently more than 100 in amniotes (Blackburn, '92), requiring close contact of maternal and embryonic tissue to form a placenta to facilitate gas and water exchange (Mossman, '37; Thompson and Speake, 2006). Ancestral amniotes provided nourishment to embryos in the form of egg yolk (lecithotrophy) and this mode of nutrition is the sole source of pre‐natal nutrients in most lineages of viviparous amniotes (Thompson et al., 2000). A rare adaptation in some viviparous lineages is the evolution of a placenta with a nutritive function, where embryos are less reliant on ovulated yolk reserves and rely
Grant sponsor: ARC Discovery. Conflicts of interest: None. Correspondence to: Oliver W. Griffith, School of Biological Sciences, Heydon Laurence Building A08, University of Sydney, NSW 2006, Australia. E‐mail:
[email protected] Received 3 April 2013; Revised 11 June 2013; Accepted 14 July 2013 DOI: 10.1002/jez.b.22526 Published online 12 August 2013 in Wiley Online Library (wileyonlinelibrary.com).
© 2013 WILEY PERIODICALS, INC.
466 on the transport of nutrients from the mother across the placenta during pregnancy (placentotrophy). Placentotrophy is most pronounced in eutherian mammals but has evolved independently in at least four additional lineages, each belonging to the skink family of lizards (Squamata: Scincidae) (Blackburn and Flemming, 2009). Although viviparity may have evolved under different selection pressures and from different evolutionary backgrounds in mammals and squamates (Blackburn, 2006), the molecular mechanisms that facilitate the function of the placenta are constrained evolutionarily by the availability of proteins already encoded in the genome of the oviparous ancestor. Although gene duplication and the introduction of functional genes by retro‐transposons have been involved in mammalian placental evolution, the majority of genes involved in placental function, for example, nutrient transport, in mammals are performed by proteins encoded by genes that have origins deep in vertebrate evolutionary history (Rawn and Cross, 2008; Dupressoir et al., 2012). Examining the multiple origins of viviparity and placentotrophy in reptiles and for comparison with mammals would allow the identification of common constraints, potential areas of genetic redundancy and for lineage specific effects that contribute to the evolution of complex structures to be identified. To address this issue, we focused on lipids to identify potential molecular mechanisms of nutrient transport because lipids are the main energy source for developing reptilian embryos and because several viviparous squamate lineages transport substantial lipid across the placenta (Speake et al., 2004; Thompson and Speake, 2006). The southern grass skink (Pseudemoia entrecasteauxii) is a placentotrophic skink found in southeastern Australia (Hutchinson and Donnellan, '92). It is highly placentotrophic with the placenta contributing approximately 60% of the lipids obtained before birth (Speake et al., 2004). Pregnancy in P. entrecasteauxii is facilitated by the formation of two placentae that form at approximately stage 30 of embryonic development (Stewart and Thompson, '96; Stewart and Thompson, 2003), while embryonic tissue is non‐invasive (Griffith et al., 2013). At the embryonic pole of the egg, the chorioallantoic membrane is in direct contact with the uterine tissue forming the chorioallantoic placenta, while at the abembryonic pole the yolk sac is in direct contact with the uterine tissue forming the yolk‐sac placenta. The formation of these two placentae coincides with an increase in the growth rate of the embryos (Itonaga et al., 2012), and it is expected that lipid transport increases through pregnancy as growth rate increases. Once formed, both the chorioallantoic and yolk‐sac placentae are retained through pregnancy, with both regions having cellular specializations that presumably have different functions. It is suspected that apocrine secretion in the yolk‐sac placenta is the most important lipid transport route (Adams et al., 2005; Stewart et al., 2006; Biazik et al., 2009), but there is no direct evidence to support that hypothesis. The major mechanism for inter‐tissue lipid transport in vertebrates is via lipoproteins in the blood (Havel, '75; Herrera et al., 2006), which release free fatty acids into tissues, when enclosed tri‐glycerides are hydrolyzed (Duttaroy, 2009). Lipoprotein J. Exp. Zool. (Mol. Dev. Evol.)
GRIFFITH ET AL. lipase is the predominant lipase responsible for the hydrolysis of tri‐ glycerides in peripheral tissues including adipose tissue, muscle heart, and the brain in vertebrates (Merkel et al., 2002) and plays an active role in the uptake of lipids by the placenta in mammalian systems (Ramsay et al., '91; Bonet et al., '92; Forde et al., 2010). We investigate the role of LPL through pregnancy in P. entrecasteauxii by measuring the spatial and temporal patterns of LPL expression in the uterine tissue of the placenta. We tested if LPL gene expression is correlated with timing of lipid transport through pregnancy in P. entrecasteauxii by measuring LPL expression through four stages of reproduction. We also measured LPL gene expression in the uterine tissue of the chorioallantoic and yolk‐sac placenta to identify which placenta is most likely responsible for lipid transport in P. entrecasteauxii.
METHODS Tissue Collection and Storage Gravid female P. entrecasteauxii (n ¼ 22) were collected in Kanangra Boyd National Park, NSW, Australia in November– December 2011. Lizards were housed individually and fed three times weekly on crickets dusted with calcium gluconate powder and mealworms. Once lizards had reached the appropriate period of the reproductive cycle, they were euthanized by injection with 0.1 mL of sodium pentobarbital (6 mg/mL). To collect tissues from non‐pregnant individuals we collected pregnant lizards from the wild and housed them through pregnancy and for an additional two months after they gave birth before processing. The egg chamber of the uterus in gravid females was excised and cut longitudinally to remove the egg. In non‐gravid females, egg chambers constituting stretched regions of the uterus, were excised individually. To examine uterine tissue belonging to the chorioallantoic placenta and omphaloplacenta separately, the uterus was cut along the boundary of the chorioallantoic membrane and yolk sac prior to removing the egg. After excision, uterine tissues were snap‐frozen in liquid nitrogen, stored in RNAlater or embedded in optimal cutting temperature compound (OCT) and subsequently stored at 80°C. After tissue processing, embryos were staged using the 40 stage staging scheme for the lizard, Zootoca vivipara (Dufaure and Hubert, '61). RNA Extraction and Purification Tissue fixed in OCT was extracted by cutting away the OCT on dry ice and melting any remaining OCT in Buffer RLT (37°C). Once tissue was removed from OCT, RNA was extracted using the same methods as snap‐frozen tissue. To extract RNA, tissue was macerated using a mechanical homogenizer in 600 mL of Buffer RLT (Qiagen, Venlo, Netherlands) then homogenized using a QIAshredder spin column (Qiagen). Total RNA was then extracted using the RNeasy Mini Kit (Qiagen). Extracted RNA was treated with Amplification Grade DNase 1 (Sigma‐Aldrich, St. Louis, Missouri, USA).
PLACENTAL LIPOPROTEIN LIPASE GENE EXPRESSION Quantitative RT PCR Lipoprotein lipase gene expression was measured in whole uterine tissue of females across four stages of the reproductive cycle, non‐ pregnant (n ¼ 6), early pregnancy (embryonic stages 1–25, n ¼ 6), mid pregnancy (embryonic stages 31–33, n ¼ 5), and late pregnancy (embryonic stages 36–38, n ¼ 5). In addition, lipoprotein lipase gene expression was measured separately in the uterine tissue belonging to the chorioallantoic and omphaloplacenta of mothers in the second half of pregnancy (embryonic stages 32–37, n ¼ 5). For each sample, high quality RNA (Bioanalyzer RIN >5) was extracted and then reverse transcribed with the QuantiTect Reverse Transcription Kit (Qiagen). LPL gene expression was normalized against the expression of b‐actin mRNA (ACTB), hypoxanthine phosphoribosyltransferase 1 mRNA (HPRT1), and 18S ribosomal RNA (18S rRNA). See Supplementary material for complete reaction conditions, primer sequences, and qPCR verification. We compared the normalized uterine LPL expression between stages of the reproductive cycle (as defined in the experimental methods) using a one‐way analysis of variance (ANOVA). Data were log10 transformed to give a non‐significant difference in variance between reproductive stages by Levene's test. Multiple pairwise comparisons were made between periods of
Figure 1. Temporal pattern of lipoprotein lipase gene expression in the uterus of P. entrecasteauxii through pregnancy. Mean relative LPL gene expression in uterine tissue for non‐pregnant (n ¼ 6), early (n ¼ 6), middle (n ¼ 5), and late pregnant (n ¼ 5) Pseudemoia entrecasteauxii. Expression data are normalized to the geometric mean of B‐actin and HPRT1 expression. Reproductive stage is significantly different (one‐way ANOVA on log10 transformed data (f3,21 ¼ 5.6, P < 0.01). Tukey's test for multiple pair wise comparisons show that late reproductive lizards have significantly greater LPL mRNA present in utero than non‐reproductive or early pregnant lizards (P < 0.05).
467 the reproductive cycle using Tukey's test. Chorioallantoic and omphalo‐placental uterine samples were collected in pairs so the data were analyzed using a paired sample t‐test. The data were log10 transformed to make the variances equal between groups. Statistical analyses were performed with SPSS (version 14.0.0, SPSS, Inc.).
RESULTS Reproductive stage has a significant effect on LPL gene expression using a one‐way analysis of variance (f3,21 ¼ 5.6, P < 0.1) with late pregnant lizards having 5.1‐ and 3.3‐fold greater LPL gene expression than non‐pregnant and early pregnant individuals respectively using Tukey's test for multiple pair wise comparisons (P < 0.05, Fig. 1). The omphaloplacenta had a mean 10‐fold greater LPL gene expression than the chorioallantoic placenta when a paired t‐test was conducted on log10 transformed data (t4 ¼ 4.773, P < 0.01, Fig. 2).
DISCUSSION Strong Support for the Role of LPL in Placental Lipid Transport Given that LPL shows a distinct pattern of increasing expression in the uterus of P. entrecasteauxii and that this pattern correlates with expected rates of increase in lipid transport in the second half of pregnancy, LPL is a good candidate for a gene involved in lipid
Figure 2. Spatial patterns of lipoprotein lipase gene expression in the uterus of P. entrecasteauxii. Relative LPL expression in the uterine tissue of the chorioallantoic placenta (CA) and omphaloplacenta (OM) (n ¼ 5) in Pseudemoia entrecasteauxii during the mid‐late stages of pregnancy. Expression data are normalized to the geometric mean of B‐actin, HPRT1, and 18S rRNA expression. The omphaloplacenta shows significantly greater LPL gene expression than the chorioallantoic placenta (paired students t‐ test on log10 transformed data (t4 ¼ 4.773, P < 0.01). J. Exp. Zool. (Mol. Dev. Evol.)
468
GRIFFITH ET AL.
Figure 3. Hypothesis for the molecular mechanisms of placental lipid transport in Pseudemoia entrecasteauxii. Lipids are carried to the blood vessels in the uterine tissue of the yolk‐sac placenta in the form of lipoproteins. Once in the blood vessels, triglycerides contained within the lipoproteins are hydrolyzed releasing fatty acids. Fatty acids are taken up by the endothelial cells via diffusion or with the aid of an additional protein. Once in the endothelium, the lipid is carried to the apical surface of the uterine epithelium where it is packaged into vesicles. Lipid transport through each cell layer and in budding vesicles is not expected to be solely in the form of fatty acids, as conversions may occur as part of the trafficking process.
transport in this region. This pattern of LPL expression was not observed in the viviparous lizard Chalcides ocellatus, presumably because C. ocellatus lacks significant placentotrophy (Blackburn, '92; Brandley et al., 2012). There is strong evidence that LPL has a role in lipid mobilization in the placentation of mammals, LPL is significantly expressed in the syncytiotrophoblast of humans (Lindegaard et al., 2005) and the endometrium of cows (Forde et al., 2010) and pigs (Ramsay et al., '91). Given the functions of lipoprotein lipase protein and its clear patterns of expression, we propose that lipoprotein lipase is present in the apical surface of endothelial cells in the uterus where it aids in the absorption of lipids from the blood supply by the hydrolysis of tri‐ glycerides in lipoproteins, releasing free fatty acids. This hypothesis could be tested by the localization of LPL to the apical surface of uterine blood vessels, as it is in mammals (Lindegaard et al., 2005), using in situ hybridization techniques. We postulate that fatty acids are taken up by the endothelium of the blood vessels by diffusion or the aid of fatty acid transport proteins (Duttaroy, 2009), and are transported to the uterine epithelium, likely involving fatty acid binding proteins as occurs in mammals J. Exp. Zool. (Mol. Dev. Evol.)
and could be tested using similar methods (Ramsay et al., '91; Forde et al., 2011). At the uterine epithelium lipids are packaged into vesicles and released into the uterine lumen for absorption by the embryonic tissue, which has been observed using electron microscopy (Adams et al., 2005). We summarize the proposed mechanism of uterine lipid transport in Figure 3.
The Yolk‐Sac Placenta and Nutrient Transport These results strongly support the hypothesis that the yolk‐sac placenta is the most likely route for lipid transport in the placenta of P. entrecasteauxii. Lipid transport across the yolk‐sac placenta likely occurs via apocrine secretions, which have been observed as budding vesicles in P. entrecasteauxii under electron microscopy (Adams et al., 2005). Moreover, the apical surface of the uterine epithelium has extensive lysosomal systems, which break down macromolecules potentially for transport to the embryo (Biazik et al., 2009). The ability of the uterus to secrete nutrients is complemented by the ability of the yolk sac to absorb complex organic molecules, including dextran (Stewart et al., 2006). The
PLACENTAL LIPOPROTEIN LIPASE GENE EXPRESSION yolk‐sac placenta is unlikely to be limited to transporting lipids alone, because packaged vesicles require the inclusion of specific packaging proteins such as clathrin and these proteins, if not returned to the mother are a source of protein transfer from mother to embryo (Schmid, '97). Transport of the amino acid leucine across the placenta has been correlated with the development of the yolk‐sac splanchnopleure in P. entrecasteauxii (Itonaga et al., 2012), which suggests that leucine transport occurs concurrently with lipids in the yolk‐sac placenta. Development of the yolk‐sac splanchnopleure is, however, correlated with the formation and development of the chorioallantoic placenta (Stewart and Thompson, '96) and therefore future work is necessary to determine the routes of protein or amino acid transport in P. entrecasteauxii.
ACKNOWLEDGMENTS Funding was from ARC Discovery grants to M.B.T., and K.B. All animal work was conducted under University of Sydney Animal Ethic approval (L04/10‐2010/1/5427 and L04/11‐2011/3/5636). Lizards were collected under New South Wales National Parks and Wildlife Licence to M.B.T. (SL100401). We thank Tommy Wallace and Peter Street with fieldwork assistance. RNA integrity analysis was carried out in the Bosch Molecular Biology Facility at the University of Sydney.
LITERATURE CITED Adams SM, Biazik JM, Thompson MB, Murphy CR. 2005. Cyto‐ epitheliochorial placenta of the viviparous lizard Pseudemoia entrecasteauxii: a new placental morphotype. J Morphol 264:264–276. Biazik JM, Thompson MB, Murphy CR. 2009. Lysosomal and alkaline phosphatase activity indicate macromolecule transport across the uterine epithelium in two viviparous skinks with complex placenta. J Exp Zool 312B:817–826. Blackburn DG. 1992. Convergent evolution of viviparity, matrotrophy, and specializations for fetal nutrition in reptiles and other vertebrates. Am Zool 32:313–321. Blackburn DG. 2006. Squamate reptiles as model organisms for the evolution of viviparity. Herpetol Monogr 20:131–146. Blackburn DG, Flemming AF. 2009. Morphology, development, and evolution of fetal membranes and placentation in squamate reptiles. J Exp Zool 312B:579–589. Bonet B, Brunzell JD, Gown AM, Knopp RH. 1992. Metabolism of very‐ low‐density lipoprotein triglyceride by human placental cells: the role of lipoprotein lipase. Metabolism 41:596–603. Brandley MC, Young RL, Warren DL, Thompson MB, Wagner GP. 2012. Uterine gene expression in the live‐bearing lizard, Chalcides ocellatus, reveals convergence of squamate reptile and mammalian pregnancy mechanisms. Genome Biol Evol 4:394–411. Dufaure JP, Hubert J. 1961. Table de développement du lézard vivipare Lacerta (zootoca) vivipara Jacquin. Arch Anat Microsc Morph Exp 50:309–328.
469 Dupressoir A, Lavialle C, Heidmann T. 2012. From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation. Placenta 33:663–671. Duttaroy AK. 2009. Transport of fatty acids across the human placenta: a review. Prog Lipid Res 48:52–61. Forde N, Spencer TE, Bazer FW, Song G, Roche JF, Lonergan P. 2010. Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium. Physiol Genomics 41:53–62. Forde N, Beltman ME, Duffy GB, et al. 2011. Changes in the endometrial transcriptome during the bovine estrous cycle: effect of low circulating progesterone and consequences for conceptus elongation. Biol Reprod 84:266–278. Griffith OW, Van Dyke JU, Thompson MB. 2013. No implantation in an extra‐uterine pregnancy of a placentotrophic reptile. Placenta 34:510–511. Havel RJ. 1975. Lipoproteins and lipid transport. Adv Exp Med Biol 63:37–59. Herrera E, Amusquivar E, López‐Soldado I, Ortega H. 2006. Maternal lipid metabolism and placental lipid transfer. Horm Res 65: 59–64. Hutchinson MN, Donnellan SC. 1992. Taxonomy and genetic‐variation in the australian lizards of the genus Pseudemoia (Scincidae, Lygosominae). J Nat Hist 26:215–264. Itonaga K, Wapstra E, Jones SM. 2012. A novel pattern of placental leucine transfer during mid to late gestation in a highly placentotrophic viviparous lizard. J Exp Zool 318:308–315. Lindegaard MLS, Olivecrona G, Christoffersen C, et al. 2005. Endothelial and lipoprotein lipases in human and mouse placenta. J Lipid Res 46:2339–2346. Merkel M, Eckel RH, Goldberg IJ. 2002. Lipoprotein lipase: genetics, lipid uptake, and regulation. J Lipid Res 43:1997–2006. Mossman H. 1937. Comparative morphogenesis of the fetal membranes and accessory uterine structures. Washington, DC: Carnegie Institution of Washington Publication. p 129–246. Ramsay TG, Karousis J, White ME, Wolverton CK. 1991. Fatty acid metabolism by the porcine placenta. J Anim Sci 69: 3645–3654. Rawn SM, Cross JC. 2008. The evolution, regulation, and function of placenta‐specific genes. Annu Rev Cell Dev Biol 24:159–181. Schmid SL. 1997. Clathrin‐coated vesicle formation and protein sorting: an integrated process. Annu Rev Biochem 66: 511–548. Speake BK, Herbert JF, Thompson MB. 2004. Evidence for placental transfer of lipids during gestation in the viviparous lizard, Pseudemoia entrecasteauxii. Comp Biochem Phys A 139A: 213–220. Stewart JR, Thompson MB. 1996. Evolution of reptilian placentation: development of extraembryonic membranes of the Australian scincid lizards, Bassiana duperreyi (oviparous) and Pseudemoia entrecasteauxii (viviparous). J Morphol 227:349–370.
J. Exp. Zool. (Mol. Dev. Evol.)
470 Stewart JR, Thompson MB. 2003. Evolutionary transformations of the fetal membranes of viviparous reptiles: a case study of two lineages. J Exp Zool 299A:13–32. Stewart JR, Thompson MB, Attaway MB, Herbert JF, Murphy CR. 2006. Uptake of dextran‐fitc by epithelial cells of the chorioallantoic placentome and the omphalopleure of the placentotrophic lizard, Pseudemoia entrecasteauxii. J Exp Zool 305A:883–889. Thompson MB, Speake BK. 2006. A review of the evolution of viviparity in lizards: structure, function and physiology of the placenta. J Comp Physiol 176B:179–189.
J. Exp. Zool. (Mol. Dev. Evol.)
GRIFFITH ET AL. Thompson MB, Stewart JR, Speake BK. 2000. Comparison of nutrient transport across the placenta of lizards differing in placental complexity. Comp Biochem Phys 127A:469–479.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher's web‐site. Table S1. Primers used in quantitative reverse transcription PCR.
