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Two Stages of Increased IgA Transfer During Lactation in the Marsupial, Trichosurus vulpecula (Brushtail Possum)

This information is current as Frances M. Adamski and Jerome Demmer of September 23, 2021. J Immunol 1999; 162:6009-6015; ; http://www.jimmunol.org/content/162/10/6009 Downloaded from References This article cites 35 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/162/10/6009.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Two Stages of Increased IgA Transfer During Lactation in the Marsupial, Trichosurus vulpecula (Brushtail Possum)1,2

Frances M. Adamski3 and Jerome Demmer

The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via

the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period Downloaded from and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation. The Journal of Immunology, 1999, 162: 6009–6015.

arsupial young are born after a short gestation, which in the colostrum (7). For marsupials, little is known about Ig trans- in the possum is only 17 days (1). Their early pouch- fer via the placenta or mammary gland. In the newborn of several life is considered to be an external gestation as many marsupials, no Igs were detected in their serum until they had M http://www.jimmunol.org/ of the developmental changes that occur in utero for eutherian suckled for the first time, suggesting that in these species there is mammals take place during this time. In particular, lymphoid or- only postnatal transfer of Igs (8–10). In contrast, Igs have been gans of the marsupial neonate are immature and pouch young are detected in the serum of newborn of the tammar wallaby (Macro- unable to elicit an immune response for some time after birth (2, pus eugenii) before suckling (11). The transfer of Igs in utero has 3). Lactation in the brushtail possum extends for ϳ200 days, dur- not been investigated for the possum. It has been shown that new- ing which milk composition and the pouch young’s behavior born possums are unable to mount a humoral response for at least changes. In early lactation (first 80 days), the marsupial young is 14 days after birth (12). Thus postpartum transfer of both IgG and confined to the pouch where it remains attached to the nipple, IgA is likely to be important for immune protection of the possum receiving milk of unique protein, carbohydrate, mineral, and fat young. A study of three brushtail possums demonstrated that Igs by guest on September 23, 2021 composition (4–6). During the switch phase (days 80–120 of lac- ingested by 50- and 98- but not 145-day-old pouch young were tation), the milk composition changes to become similar in com- subsequently detected in their serum (13). This suggests that, al- position to that of eutherian mammals. With the change in milk though older pouch young can mount their own immune response, composition, growth of the pouch young increases, and they un- they remain receptive to the passive immunity offered by their dergo changes such as fur growth, their activity increases, suckling mothers milk, at least into the switch phase of lactation. is more intermittent (after day 80), and they begin to leave the Passively transferred IgA functions to protect mucus mem- pouch (after day 115). branes (respiratory and gastrointestinal) of the newborn. In euth- At birth, eutherian young are immunologically naive and they erian mammals, lymphocytes associated with mammary gland ep- receive immune protection by ingestion of Igs secreted into the ithelium secrete IgA as a dimer joined by the J chain (14). Dimeric colostrum until they are capable of mounting their own immune IgA is transported across the epithelial cells into the milk by the response. In mammals such as humans, where IgG is transferred polymeric Ig receptor (pIgR)4 and is released at the apical mem- predominately via the placenta, IgA is the major Ig transferred via brane by proteolytic cleavage of the extracellular domain (secre- the colostrum. In contrast, when little or no IgG is transferred tory component (SC)) (15). It has been suggested that the SC pro- before birth, as seen in the cow, more IgG than IgA is transferred tects dimeric IgA from proteolytic cleavage in mucosal environments (16). The possum IgA heavy chain (C␣) DNA se- Dairy Science Group, AgResearch, Ruakura Research Centre, Hamilton, New Zea- quence was published recently by Bevlov et al. (17). land Immune transfer to the possum neonate via the milk has not Received for publication October 7, 1998. Accepted for publication February been studied in great detail. In this work, we have cloned and 26, 1999. characterized the first marsupial homologues of the pIgR and J The costs of publication of this article were defrayed in part by the payment of page chain genes. Analysis of the expression pattern of the pIgR, J charges. This article must therefore be hereby marked advertisement in accordance chain, and C␣ mRNA suggests that there are two stages of in- with 18 U.S.C. Section 1734 solely to indicate this fact. creased IgA transfer during lactation in the possum, after birth and 1 This work was supported by Ministry of Agriculture, and Forestry Policy, New Zealand, under contract PBC/09. during the switch phase. 2 All sequences reported have been deposited into the GenBank database, accession nos. AF091138 (polymeric Ig receptor), AF091138 (J chain), and AF091139– AF091141 (IgA heavy chain). 3 Address correspondence and reprint requests to Dr. Frances M. Adamski, Dairy 4 Abbreviations used in this paper: pIgR, polymeric Ig receptor; IgA heavy chain, C␣; Science Group, AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, SC, secretory component; RACE, rapid amplification of cDNA end; CDR, comple- New Zealand. mentarity-determining region.

Materials and Methods with 32P-radiolabeled cDNA probes in Church and Gilbert hybridization ϫ RNA purification solution (22) following 30 min prehybridization. Blots were washed in 2 SSC/0.1% SDS at 55°C, then 1ϫ SSC/0.1% SDS for 20 min each. Blots Mammary gland total RNA. Mammary glands were collected as described were exposed to Kodak XAR-5 film (Rochester, NY) at Ϫ80°C with two previously (6), and the date of lactation was based on the size of the pouch intensifying screens. The possum 18S rRNA probe is an unpublished DNA young (18). The frozen mammary tissues were ground under liquid nitro- fragment (400 bp) amplified by RT-PCR (J. Demmer, GenBank gen by mortar and pestle, and RNA was extracted using the guanidinium AF089722). acid phenol chloroform method (19) with the modification of reducing the recommended amount of tissue per volume of reagent by one half. Results White blood cell total RNA. RBC were lysed by the addition of 12 ml of Isolation of possum pIgR gene lysis solution (150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) to 4.5 ml of heparin-treated possum blood, incubating on ice for 20 min. White A 156-bp DNA fragment corresponding to the possum pIgR was blood cells were harvested by centrifugation and washed with 1ϫ PBS amplified by RT-PCR using mammary gland total RNA (28 days before RNA isolation as described above. lactation) and oligonucleotide primers designed to conserved re- RT-PCR and 5Ј rapid amplification of cDNA end (RACE) gions of the pIgR coding sequence. This fragment was used to screen an early lactation possum mammary gland cDNA library, Random or oligo-dT primed cDNA was generated using the Superscript and 12 positive cDNA clones were isolated. Of these, three re- preamplification system (Life Technologies, Gaithersburg, MD) as per the manufacturer’s instructions. PCR was performed using Platium Taq (Life combinants containing different-sized inserts were characterized Technologies) or Taq DNA polymerase (Boehringer Mannheim, Mann- by DNA sequencing. All three recombinants contained pIgR se- heim, Germany). The 5Ј RACE was performed as described by Life Tech- quence, and the DNA sequence of the largest cDNA clone (2066 nologies using Superscript II reverse transcriptase and RNase H from Life bp) was determined for both DNA strands. This recombinant con- Downloaded from Technologies and terminal transferase kit and RNase T1 from Boehringer Mannheim. Primer-specific cDNA was purified using the BRESAclean tained the majority of the coding sequence (from nt 782; Fig. 1). DNA purification kit (Bresatec, Thebarton, Australia). Upstream primers, The remainder of the coding and 5Ј untranslated sequences were abridged anchor primer and abridged universal amplification primer, used obtained by the 5Ј RACE procedure. The possum pIgR is a 733-aa in the PCR reactions are as described by Life Technologies. All PCR prod- polypeptide including the 17-aa signal peptide. It shares 44–48% ucts were cloned into pGEM-Teasy (Promega, Madison, WI), and at least amino acid identity to human, murine, bovine, and rat pIgR se- two clones of each were DNA sequenced. pIgR. RT-PCR was performed with 5Ј primer (GAA TTC CTA GCA quences, which is lower than that observed between these species http://www.jimmunol.org/ CCA RTA CCA NCC YTC) and 3Ј primer (GGA TCC TAY TGG TGY (56–80%) with the exception of the rabbit pIgR sequence, which AAR TGG). PCR conditions were annealing at 37°C for 3 cycles, then has low homology with the possum pIgR (40%) as well as euth- Ј 55°C for 27 cycles with 60-s extensions. The 5 RACE was performed with erian pIgR sequences (43–47%). GSP1 (CTG GCA TAG AAA CTT GG), GSP2 (AGG GCA CAG TTC AAG GTC AC), and GSP3 (AGG CTC CAG CAA CTT TAA GTC). PCR The marsupial pIgR sequence was compared with that of euth- conditions for PCR1 were annealing at 50°C for 10 cycles then 55°C for 20 erian species to determine whether or not important functional do- cycles with 4-min extensions, and for PCR2 were annealing at 57°C for 30 mains have been conserved. The possum pIgR contains all five cycles with 2.5-min extensions. extracellular Ig-like domains, including the cysteine residues in- Ј J chain. RT-PCR was performed with 5 primer (GGA TCC GYT GAY volved in intramolecular disulfide bonds (Fig. 1). The 23-aa resi- AAY AAR TG) and 3Ј primer (GGA TTC CTA RTC AGG RTA), an- by guest on September 23, 2021 nealing at 37°C for 4 cycles, 45°C for 4 cycles, then 50°C for 26 cycles, due polymeric Ig-binding domain, located in the first Ig-like do- with 90-s extensions. RT-PCR analysis of J chain mRNA expression was main, is involved in the initial noncovalent interaction with performed with 5Ј primer (GAG GCA AGA TGA AGA GAT CT) and 3Ј polymeric Ig (23). In the case of dimeric IgA, this is followed by primer (GTG CAG TTT CAA GAT GGA AG). PCR conditions were the formation of a disulfide bridge between one of the C␣ chains annealing at 58°C for 25 cycles with 80-s extensions. IgA. RT-PCR was performed with 5Ј primer (GAG GCA AGA TGA and the fifth Ig-like domain of the pIgR (24). In the possum pIgR, AGA GAT CT) and 3Ј primer (GTG CAG TTT CAA GAT GGA AG) with both the pIg-binding domain (Fig. 2A) and the cysteine residues four PCR cycles each at 65°C, 60°C, 57°C, 53°C, and 50°C, then 10 cycles involved in the formation of the covalent interaction are conserved at 60°C with 2-min extensions. For 5Ј RACE, primers GSP1 (CAA GCA (Fig. 1, circles). This suggests that the mechanism for receptor-Ig GCC TAG GAC TA), GSP2 (AGT CTC CGA GTA GAA TGA G), and interaction has been conserved in marsupials. GSP3 (GGA GAC AGT CAC CAT G) were used as described for the pIgR. The possum pIgR transmembrane domain is also highly conserved within the 10-aa core (Fig. 2B, boxed region). It has been Library screening suggested that this region may be involved in transducing a signal, The EcoRI-EcoRI DNA fragments from clones containing pIgR and J generated by ligand binding, to the cytoplasmic membrane leading chain RT-PCR products were purified and used to generate 32P-radiola- first to endocytosis and then transcytosis of the receptor to the beled probes with the Rediprime random primer labeling kit (Amersham, apical membrane (25). The region of lowest sequence conservation Aylesbury, U.K.). These were used to screen the possum early lactation mammary gland cDNA library (20) using methodology previously de- was in the linker region (domain VI; Fig. 1) connecting the Ig-like scribed (21). Recombinants were purified to single plaques and the internal and transmembrane domains. This region is the most variable be- plasmid (pBS; Stratagene, La Jolla, CA) was excised with ExAssist helper tween all the pIgR sequences with respect to size and sequence (25). phage (Stratagene) following recommended procedures. The resulting plas- The pIgR cytoplasmic domain contains signaling sequences for mids were characterized by DNA sequencing. basolateral targeting, endocytosis, and transcytosis (26, 27), and DNA sequencing this region of the possum pIgR is highly conserved with eutherian sequences (68–78% identity). Signals required for basolateral tar- DNA sequencing reactions were conducted by the DNA Sequencing Unit (University of Waikato, Hamilton, New Zealand) using an automated DNA geting of the de novo pIgR reside in a region proximal to the sequencer (Applied Biosystems, Foster City, CA). DNA sequence data was transmembrane domain. Three amino acid residues have been pre- collated using the SEQMAN II program of the Lasergene software package viously defined as being important for basolateral signaling in the (DNASTAR, Madison, WI). rabbit pIgR (Fig. 2C, underlined sequence) (28). However, these Northern blotting three residues are not conserved in the possum pIgR, suggesting that either the possum receptor uses a different mechanism for Total RNA (6 ␮g) was denatured in the presence of formamide and formaldehyde and then resolved by gel electrophoresis in 1.5% agarose, 0.6 M basolateral targeting or that the basolateral signaling sequence is formaldehyde gels (22). RNA was transferred to Hybond N membrane larger than that previously characterized. Interesting, amino acids (Amersham) by capillary blotting. Blots were hybridized overnight at 55°C immediately distal to these residues are highly conserved between The Journal of Immunology 6011 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. Possum pIgR: DNA and deduced amino acid sequence. Nucleotide and amino acid numbers are indicated to the right. The signal peptide is italicized, and the transmembrane region is in bold. Boundaries of the Ig-like domain and the domain linking these and the transmembrane domain are numbered (I–V and VI, respectively). Conserved cysteines are indicated by asterisks, and cysteines involved in bringing about the covalent bond between the ﬁrst of these cysteines and the Ig heavy chain are circled. The basolateral targeting (baso) and endocytosis (endo) signal sequences are indicated. GenBank accession no. AF091137. 6012 IgA TRANSFER DURING POSSUM LACTATION

(18 days lactation) and oligonucleotide primers designed to conserved regions of the J chain. This DNA fragment was used as a probe to isolate three cDNA clones from the early lactation possum mammary gland cDNA library. Two of these contained the entire J chain coding sequence, and the DNA sequence was determined for the largest cDNA clone (1620 bp; Fig. 3). The possum J chain is 160 aa in length and has a leader peptide of 22 aa. The possum J chain amino acid sequence has 63–68% identity with eutherian sequences, which is similar to that observed between eutherian species (67–80%). The cysteine residues that form intra- and intermolecular disulﬁde bonds are conserved (Fig. 3) (32).

Cloning of the possum C␣ variable domain The possum C␣ variable and constant domains were ampliﬁed by 5Ј RACE and RT-PCR, respectively, using total RNA from possum white blood cells and oligonucleotide primers based on the

known possum C␣ constant domain nucleotide sequence (17). The Downloaded from DNA sequence was determined for two clones containing the variable domain, and a high degree of amino acid variation was found in the complementarity-determining regions (CDR1, CDR2, and CDR3-D-J) (V-1 and V-2; Fig. 4). As expected, there were also amino acid differences in the framework regions and in the leader

peptide sequence. The CDR3-D-J region is assembled by recom- http://www.jimmunol.org/ bination of the germline V (variable), D (diversity), and J (joining) segments resulting in the differences in amino acid identity and length seen in this region. The amino acid sequence of the constant domains (CH1–CH3) of the New Zealand possum (C-2) differed from that obtained from the Australian possum (C-1) at three amino acids: Asn144 to Asp, Arg193 to Gly, and Asn399 to Lys (Fig.

FIGURE 2. Interspecies homology among pIgR amino acid sequences 4). A recent study of the VH repertoire of the marsupial Monodel-

in regions with known function. Sequences compared include (species, phis domestica (South American short-tailed opossum) demon- by guest on September 23, 2021 GenBank accession no.) pIgR of human (Homo sapiens, P01833), bovine strated the presence of two VH families that are phylogenetically (Bos taurus, L04797), rat (Rattus norvegicus, P15083), mouse (Mus mus- most similar to the group III family in vertebrates (33). The vari- culus, U06431), rabbit (Oryctolagus cuniculus, P01832), and possum. A, able domains of the possum C␣ are most homologous to the Ig-binding domain. B, Transmembrane domain; the highly homologous 10-aa core sequence is boxed. C. Basolateral targeting domain; amino acid MdoVH1 family (results not shown). residues shown to have important function in basolateral targeting are underlined (rabbit sequence), and the region most highly homologous between all species is boxed. D, Least conserved region of the cytoplasmic Expression of the IgA, J chain, and pIgR mRNA in the domain. E, Endocytosis domain; the region of highest homology is boxed. mammary gland during lactation Dimeric IgA is expressed by lymphocytes associated with the mammary gland epithelium. The expression pattern IgA in the all the pIgR sequences (Fig. 2C, boxed), suggesting that they may mammary gland during lactation was determined by analysis of form part of the basolateral targeting signal. C␣ and J chain mRNA expression. Northern blots of total RNA The central region of the cytoplasmic domain, which has been extracted from mammary glands at different stages of lactation implicated to have a role in transcytosis, is the least conserved were hybridized with the J chain and IgA probes (Fig. 5A). The region between all species (Fig. 2D) (26). The only amino acid expression of both these mRNAs was increased for two short pe- shown to have a direct role in transcytosis is conserved in the riods during lactation: at the beginning of lactation (days 1.5–6) possum sequence (Ser642; Fig. 2C), which lies outside of this re- and, unexpectedly, again in the switch phase (days 106, 110, and gion (29). Phosphorylation of this serine in the rabbit pIgR stim- 115). Expression of the J chain mRNA during early lactation (days ulates transcytosis in the absence of ligand binding. Truncation of 31–94) was demonstrated using RT-PCR (Fig. 5C). This suggests the carboxyl-terminal 30 aa has been shown to affect the endocytosis of the receptor from the basolateral membrane (26). Part of that there is expression of dimeric IgA during this period of lac- this region is highly conserved in the possum (Fig. 2E, boxed tation, albeit at a low level. region) and contains amino acid residues Ser703 and Tyr711 (of the If the transfer of IgA into milk is increased for these two periods possum sequence), which in addition to Tyr646 have been shown in of lactation, then expression of the pIgR, which transports dimeric other species to be phosphorylated in response to ligand binding, IgA across epithelial cells, is also likely to be increased at these leading to endocytosis (30, 31). times. The pIgR mRNA was found to be expressed throughout lactation (Fig. 5A). Importantly, we ﬁnd that there are two periods Isolation of possum J chain gene during lactation when expression of this mRNA was increased: at A 400-bp DNA fragment corresponding to the possum J chain was the beginning of lactation (days 1.5–6) and again from the switch ampliﬁed by RT-PCR using possum mammary gland total RNA phase of lactation until the end of lactation (days 106–189). It The Journal of Immunology 6013

FIGURE 3. Possum J chain: DNA and deduced amino acid sequence. Nu- cleotide and amino acid numbers are indicated to the right. Conserved cysteine residues involved in intra- (underlined) and intermolecular (asterisk) disulphide bonds are indicated. GenBank accession no. AF091138. Downloaded from http://www.jimmunol.org/

would appear that the pattern pIgR mRNA expression coincides of these possum genes are highly conserved with those of euther- with that of the J chain and C␣, with expression being highest at ian mammals. the beginning of lactation and again from the latter part of the The mRNAs of pIgR, C␣, and J chain were found to be ex- switch phase. pressed in the mammary gland throughout lactation, suggesting that there is continuous transfer of IgA into possum milk. Expres- Discussion sion of C␣ and J chain mRNAs were dramatically increased at two by guest on September 23, 2021 This report describes the cloning and characterization of the first specific periods of lactation, which are summarized in Fig. 6. The marsupial pIgR and J chain cDNAs from the brushtail possum. The first period was at the beginning of lactation, and this would be pIgR is involved in transport of dimeric IgA across epithelial cells consistent with IgA transfer via the colostrum as seen in euther- and the J chain functions to dimerize IgA molecules (14, 15). Both ians. The second period of increased mRNA expression occurred

FIGURE 4. Possum C␣. Aligned amino acid sequences of the variable domain of two C␣ chains (V-1 and V-2), and C␣ constant regions of New Zealand (C-2) and Australian (C-1) (17) (GenBank accession no. AF027382) origin. The variable (VH) and constant (CH1–CH3) domains are indicated. The leader peptide is italicized, and the hinge sequence is underlined. CDR1, CDR2, and CDR3-D-J are boxed, including the diversity (D) and joining (J) segments. The differences in amino acid identity between the sequences are indicated in bold. GenBank accession nos. AF091139–AF091141. 6014 IgA TRANSFER DURING POSSUM LACTATION

eutherian mammals. Both periods of increased IgA transfer would occur when the pouch young are exposed to new pathogens. In- deed, there is a high rate of mortality in the possum pouch young at birth and around the switch phase (34), and increased protection at these stages would be advantageous. The presence of IgG and IgA in possum milk has not been reported, but is inferred from experiments describing passive transfer of immunity to the neonate and the presence of IgA in the milk of other marsupials (10, 11, 13, 35). We have detected both IgG and IgA in possum milk (Adamski et al., unpublished observa- tions), and although no information regarding their concentration in milk is available, it is likely that both play a role for immune protection of the pouch young. Interestingly, a dramatic rise in the IgG serum concentration was seen at the switch phase in the hill kangaroo (Macropus robustus) pouch young, subsequent to the pouch young beginning to leave the teat and before pouch exiting (36). This increase in serum Ig concentration is thought to reﬂect the pouch young’s immune response, but may also result from an

increase in immune transfer via the milk. The importance of these Downloaded from two periods of immune transfer to the survival of the possum FIGURE 5. Expression pattern of C␣, J chain, and pIgR mRNAs in the pouch young will require detailed analysis of IgA and IgG con- possum mammary gland. A, Northern blots hybridized with J chain centration in the colostrum and milk throughout lactation. (mRNA, ϳ1600 bases), C␣ (mRNA, ϳ1800 bases), and pIgR (mRNA, Colostral secretion of IgA is likely to be controlled by mam- ϳ 2900 bases) probes. The day of lactation when the mammary gland tissue motrophic hormones affecting the expression of receptors and Igs. was sampled is indicated. The apparent anomaly in J chain and C␣ ex-

In mice administration of prolactin, progesterone and estrogen re- http://www.jimmunol.org/ pression for days 113–115 is presumably due to an error in estimation of sulted in the development of the mammary epithelia with a con- the time of lactation in one of these animals. B, The J chain (top panel) and C␣/pIgR (bottom panel) Northern blots hybridized with the 18S rRNA comitant increase in the number of associated IgA-secreting probe to show relative RNA loading. C, RT-PCR analysis of J chain plasma cells and intraepithelia IgA (37). Interestingly, in marsu- mRNA expression, performed on total RNA obtained from mammary pials including the possum there are two periods when serum con- gland tissue at different days of lactation. Controls include the 113-day centration of prolactin is elevated, which coincide with increased sample processed without reverse transcriptase added (113-RT) and the dimeric IgA and pIgR expression in the mammary gland (38). PCR reaction performed without cDNA added (no template). There is a short peak before initiation of lactation and a second period of prolonged elevated serum prolactin concentration around the switch phase, increasing from approximately day 100 in the by guest on September 23, 2021 for a short period (days 106–115) toward the end of the switch possum. An alternative possibility is that dimeric IgA expression is phase (80–120 days of lactation). Expression of the pIgR mRNA stimulated locally by the introduction of novel pathogens by the was also increased at these two stages of lactation, with the level pouch young. The induction of an IgA Ab response by local chal- of expression at the second period remaining elevated to the end of lenge of the mammary gland is characteristic of eutherians and has lactation (Fig. 6). The second period of increased expression of been demonstrated in the quokka, Setonix brachyurus (35). How- these mRNAs is likely to represent a second period of enhanced ever, this latter explanation would not explain the second period of IgA transfer via the milk. This second period coincides with the increased pIgR mRNA expression. end of the external gestation period of the pouch young’s life and In summary, we have cloned and characterized the pIgR and J could be thought of as a second colostral phase. Alternatively, the chain of the brushtail possum. The expression pattern of the J timing of this second period of IgA transfer, just before pouch exit, chain, C␣, and pIgR mRNA during lactation suggests that there are suggests the intriguing possibility that this could be equivalent to two stages of increased immune transfer in the possum at the be- the placental Ig transfer that occurs during late gestation in some ginning of lactation and toward the end of the switch phase. We propose that these represent two colostral phases during lactation, the second being unique to marsupials. Acknowledgments We thank Susan Stasiuk for excellent technical assistance, Dr. Tim Day for sampling of possum blood, and Dr. Murray Grigor for helpful discussions. References 1. Sharman, G. 1962. The initiation and maintenance of lactation in a marsupial, Trichosurus vulpecula. J. Endocrinol. 25:375. 2. Ashman, R. B., and J. M. Papadimitriou. 1975. Development of the lymphoid tissue in a marsupial, Setonix brachyurus (quokka). Acta Anatomica 91:592. FIGURE 6. Comparison of dimeric IgA and pIgR mRNA expression 3. Block, M. 1964. The blood forming tissues and blood of the new born opossum (Didelphis virginiana). I. Normal development through about the one hundredth with the stage of possum lactation. The bar indicates the sequential periods day of life. Ergeb Anat. Entwicklungs Gesh. 37:237. of short gestation (dark shading) and the early, switch, and late stages of 4. Cowan, P. E. 1989. Changes in milk composition during lactation in the common lactation. Time of birth (day 0) and approximate times when intermittent brushtail possum, Trichosurus vulpecula (Marsupialia: Phalangeridae). Reprod. suckling and pouch exiting begin are indicated. The changes in the expres- Fertil. Dev. 1:325. ␣ 5. Crisp, E. A., P. E. Cowan, and M. Messer. 1989. Changes in milk carbohydrates sion pattern of IgA (C and J chain) and pIgR mRNAs throughout lactation during lactation in the common brushtail possum, Trichosurus vulpecula (Mar- are proﬁled below (not to scale). supialia: Phalangeridae). Reprod. Fertil. Dev. 1:309. The Journal of Immunology 6015

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