Short-Tailed Opossum

MISCELLANEOUS SMALL MAMMAL BEHAVIOR

DAN H. JOHNSON , in Exotic Pet Behavior, 2006

Sensory Behaviors

Short-tailed opossums are very sensitive to light and noise. Excessive ultraviolet light exposure may lead to retinal melanoma. Short-tailed opossums have well-developed olfactory systems and rely heavily on their sense of smell.

Short-tailed opossums are sensitive to high frequencies, yet relatively insensitive to sound. At 60 dB sound pressure level (SPL), the hearing of Monodelphis extends from 3.6 kHz to 77 kHz, with a range of best sensitivity from 8 to 64 kHz. They are not particularly sensitive to tones, with the lowest threshold near 20 dB SPL. 18 Ears of short-tailed opossums are thin, hairless, and subject to desiccation; the pinnae will shrivel under prolonged exposure to low humidity. 55

These opossums demonstrate less fear of new objects and investigate them more intensely than rats. 65 This may be because short-tailed opossums are nomadic and therefore routinely come in contact with objects that are new to them.

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Anesthesia and Analgesia in Other Mammals

Jeff Wyatt , in Anesthesia and Analgesia in Laboratory Animals (Second Edition), 2008

2. Short-tailed Opossum—Monodelphis domestica

The short-tailed opossum is a small marsupial (90–155 g) found throughout the forests of Brazil, Bolivia, Argentina, and Paraguay. Their gestation is of 14–15 days with postpartum attachment to nipples for 3–4 weeks ( Moore and Myers, 2006). The short-tailed opossum has a rudimentary flap of abdominal skin instead of a pouch (Johnson-Delaney, 2006). The short-tailed opossum is used in studies of exercise metabolism (Schaeffer et al., 2005), developmental anatomy and physiology (Kraus and Fadem, 1987; Robinson and Van de Berg, 1994; Stolp et al., 2005), ultraviolet radiation–induced melanoma (Robinson and Van de Berg, 1994), and cytogenetics (Kraus and Fadem, 1987). There is no orbital sinus for blood collection (Kraus and Fadem, 1987).

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Mammals

R.J. Nudo , S.B. Frost , in Evolution of Nervous Systems, 2007

3.28.3.3 Emergence of a True Motor Cortex

In eutherian species, M1 (i.e., MsI) is situated rostral to S1 (i.e., SmI) and has a parallel somatotopic organization, though modern microstimulation studies (e.g., Gould et al., 1986) have revealed that the motor map is organized in a fractionated mosaic distribution with respect to evoked joint movements, in contrast to the relatively precise topography of receptive field organization in somatosensory cortex. The neocortex of two extant marsupials, the North American opossum (D. virginiana ) and the South American gray short-tailed opossum ( Monodelphis domestica) have been studied extensively in order to determine if any evidence of a primordial motor cortex could be found rostral to S1. These more recent studies employed microelectrode stimulation and recording techniques, allowing much greater spatial resolution of somatosensory and motor maps than afforded by the surface stimulation and recording techniques used in the early 1960s. Examination of Didelphis cortex using microelectrode techniques revealed that movements can be evoked from several sites within, and some sites just rostral to S1 (Beck et al., 1996). Stimulation most often resulted in movements of the tongue, though evoked movements of body parts, including several forelimb movements, were also seen. Together with the evidence that most CS fibers originate from S1 in Didelphis, and very few originate from areas rostral to S1, the results so far seem to favor the idea that Didelphis cortex contains S1, but not a true motor cortex.

The gray, short-tailed opossum (M. domestica) may possibly represent an even earlier stage of somatosensory-motor differentiation. In this species, movements can be evoked from a large area coextensive with the somatosensory representation, as was found for Didelphis (Frost et al., 2000). However, unlike Didelphis, in which orofacial as well as proximal and distal forelimb movements were evoked, electrical stimulation in Monodelphis evoked movements restricted almost exclusively to the vibrissae, and to a lesser extent, jaw (see Somatosensory Specializations in the Nervous Systems of Manatees). In this study, three modes of stimulation were used:

1.

intracortical microstimulation (750   kΩ electrode impedance) in layer V;

2.

low-impedance depth stimulation (100–200   kΩ electrode impedance) in layer V; and

3.

bipolar surface stimulation.

Results were qualitatively similar using the different techniques, though the excitable area increased progressively with methods 2 and 3 (Frost et al., 2000). No motor representation of body parts below the level of the head was found in S1 or in more rostral areas.

These results suggest that evolutionary pressures for development of motor components in cerebral cortex may have begun long before the emergence of a true, differentiated motor cortex, possibly due to a need for more refined movements of the face (Frost et al., 2000). This putative selective pressure may have resulted in an early substrate for mediating movements of vibrissae, long before sufficient CS pathways were in place to mediate direct control of the forelimbs and hindlimbs, and long before a separate motor representation rostral to S1 was established (Figure 1). In this regard, it is important to note that Monodelphis contains almost 20 times fewer CS neurons than Didelphis, and nearly 40 times fewer than rat (Nudo et al., 1995). Thus, S1 of Monodelphis may represent a primordial condition in which a complete somatosensory map has been achieved and retained, but the motor components of S1 have not yet fully developed. In this primitive condition, the anatomical substrate required for cortical control of movements below the level of the face is not yet present.

Figure 1. Hypothetical model of evolutionary divergence in sensorimotor cortex. a, Somatomotor representation in the earliest mammals, represented by Monodelphis. A complete somatosensory (SI) representation exists. An incomplete motor representation is congruent with the sensory representation of the face (SmI). b, A complete motor representation exists and overlaps the somatosensory representation (SmI). As noted by Lende, this sensorimotor amalgam is not unlike S1 of placental mammals. A reversed map characteristic of true motor cortex is absent. c, True motor cortex has emerged, as evident from a reversed motor representation (MsI) rostral to the somatosensory representation (SmI). MsI is largely segregated from SmI, though a partial overlap exists in the hindlimb and forelimb areas. d, In primates, MsI is completely segregated from SmI. Somatosensory cortex retains some motor properties, and motor cortex retains some somatosensory properties.

Based on these and other studies, it is thought that in opossums, motor functions at the level of the cerebral cortex, if they exist, are mediated by motor components of somatosensory cortex. Further, the motor map in S1 may have developed in stages, as it is restricted to the face, or to the face and upper extremity in Monodelphis and Didelphis, respectively. The cortical organization in monotremes, however, suggests that sensorimotor differentiation may not have followed a simple linear progression. For example, using surface stimulation techniques in the prototherian echidna (Tachyglossus aculeatus), Lende (1964) found a complete motor representation overlapping the somatosensory representation. Using surface stimulation techniques in the platypus (Ornithorhynchus anatinus), Bohringer and Rowe (1977) found a partial overlap of the motor and somatosensory representations. Electrical stimulation evoked movements of the bill and forelimb, but not the hindlimb. Thus, it is possible that the motor component of S1 is more complete in monotremes than in Monodelphis opossums.

Although motor components in S1 have been found in all mammals studied to date, the issue of whether a separate primordial motor cortex rostral to S1 exists in neurologically primitive mammals is not yet clear. In early surface stimulation studies in echidna (Abbie, 1938; Goldby, 1939) evoked movements were observed in an area rostral to S1. Surface stimulation studies in platypus also resulted in evoked movements rostral to S1, including representations of the forelimb. But the area immediately rostral to S1 in monotremes (rostral field or field R) contains neurons responsive to stimulation of deep receptors (Krubitzer et al., 1995), similar to neurons in area 3a of eutherian mammals. Area 3a is considered to be a transition zone between area 3b of S1 and area 4 (M1). In eutherian mammals, movements can be evoked by microelectrode stimulation in area 3a using relatively low current levels. This raises the possibility that area 3a became differentiated from S1 prior to the emergence of a true motor cortex. Furthermore, based on electrophysiological and cytoarchitectonic criteria, a separate cortical area (the manipulation field, or field M) has been identified in both echidna and platypus (Krubitzer et al., 1995). As field M shares some cytoarchitectonic similarities with M1, it is possible that this area is a homologue of M1 in eutherian mammals (Krubitzer et al., 1995). However, at this point it is also possible that field M is a unique specialization in extant monotremes. Clearly, additional studies of its physiology and anatomy are needed to resolve this issue.

These findings of a possible M1 homologue rostral to S1 in monotremes suggest that the antecedents to a true motor cortex may have existed very early after the emergence of mammals. As evolutionary pressures for specialized motor functions (e.g., manual dexterity) grew, augmentations of the CST from a primitive to a more advanced pathway seem to have paralleled the origin of a true motor cortex.

It is not entirely clear when true motor cortex might have emerged. But it seems that somatosensory-motor differentiation occurred independently in several orders, and not just in primates. For example, a true motor cortex, as evidenced by a reversed motor representation rostral to the somatosensory representation and by movements evoked at relatively low microelectrode stimulating currents, is apparent in rats (Donoghue and Wise, 1982). Motor cortical fields have been localized to frontal cortex in other rodent species as well (Woolsey et al., 1952; Hall and Lindholm, 1974). A separate and distinct topographic pattern comprising M1 in the rat forms a rough mirror image of the S1 representation, with the hindlimb caudomedial, the face rostrolateral, and the proximal and axial representations rostromedial (Neafsey et al., 1986; Neafsey and Sievert, 1982). In rats, the separation of S1 and M1 is not complete, since some overlap has been demonstrated over most of the hindlimb representation and part of the forelimb representation (Hall and Lindholm, 1974; Donoghue and Wise, 1982; Sanderson et al., 1983). This area of overlap has features similar to both S1 and M1 cortex. The overlapping hindlimb area receives a convergence of thalamic projections from the ventrolateral nucleus and the ventrobasal complex, whereas in nonoverlapping areas these projections are segregated (Donoghue et al., 1979). Regardless of the partial overlap in rats, it appears that a true, separate motor area, distinct and separate from the S1 cortex exists in rodents, primates, and carnivores.

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Reproductive Endocrinology of Prototherians and Metatherians

Bronwyn M. McAllan , in Hormones and Reproduction of Vertebrates: Mammals, 2011

4.2 Marsupials

Control of ovarian cycles is known for a few marsupials, including the kowari (Dasyuroides byrneii), the brush-tailed possum, the stripe-faced dunnart, the grey-short-tailed opossum (all type 1 reproductive pattern), and the tammar (type 3 reproductive pattern). Plasma LH remains low for most species until behavioral estrus, when there is a sharp rise. In tammars, there is a short-lived LH surge followed one to two days later by ovulation ( Sutherland et al., 1980; Harder et al., 1985) and a similar pattern is seen in the brush-tailed possum with a short-lived LH surge coincident with behavioral estrus one day before ovulation (Shorey & Hughes, 1973; Crawford et al., 1999). In contrast, the pattern of LH secretion in the kowari rises in the first part of the ovarian cycle and shows no surge prior to ovulation (Fletcher, 1989b).

In female tammars, basal concentrations of LH are low (0.94   ng . mL−1) with pulses of low magnitude (1.3   ng . mL−1) and low frequency (Hinds, Diggle, & Tyndale-Biscoe, 1992). Nonluteal ovarian feedback is necessary for LH secretion from the pituitary, and E2 promotes the LH surge (Hinds et al., 1992b; Rudd, Short, McFarlane, & Renfree, 1999). Removal of the sucking stimulus in tammars affects LH pulse frequency but there are no seasonal differences in pulses of LH (Hinds et al., 1992b). In the brush-tailed possum, mRNA coding for LH receptors are found in the granulosa cells of the ovary, first appearing at the time of antrum formation in the theca interna, and are also present in the interstitial tissue, although the tissue does not appear to respond to LH stimulation (Eckery et al., 2002b; Haydon, Juengel, Thomson, & Eckery, 2008).

Data for the role of follicle-stimulating hormone (FSH) in the reproductive cycle of female marsupials are patchy. In tammars, the pattern of FSH release is not pulsatile (Hinds et al., 1992b). Circulating FSH does not change when the sucking stimulus of young is removed, but concentrations are higher during seasonal quiescence (Hinds et al., 1992b). Concentrations of FSH in the plasma of female tammars are low but increase around the time of estrus, coinciding with an increase in follicular development (Evans, Tyndale-Biscoe, & Sutherland, 1980). Treatment of both cycling and noncycling female tammars and brush-tailed possums with exogenous eutherian GTHs (porcine LH and FSH in females primed with pregnant mare serum gonadotropin (PMSG)) increases the number of developing follicles and ovulation sites (Glazier & Molinia, 1998; Molinia, Gibson, Smedley, & Rodger, 1998). The response is seasonal in brush-tailed possums (Glazier, 1998), but not in tammars (Molinia et al., 1998). Nonluteal ovarian tissue is essential for the negative-feedback effects on both LH and FSH secretion in the tammar (Hinds et al., 1992b). The FSH receptor (FSH-R) gene has been cloned and characterized in this species and found to share 94% amino acid similarity with human FSH-R, and is expressed in both the adult testis and ovary (Mattiske, Pask, Shaw, & Shaw, 2002). This would suggest a similar function for this gene in both marsupials and eutherians.

Specific sites for the FSH receptor include the granulosa cells of healthy follicles containing at least two complete layers of cells in brush-tailed possums (Eckery et al., 2002b). In the brush-tailed possum, expression of the FSH-R was observed in granulosa cells of follicles shortly after they had begun to grow, was limited to these granulosa cells, and was active in granulosa cells of follicles shortly after antrum formation (Eckery et al., 2002b). Similarly to eutherian mammals, multiple ovulatory follicles can be induced to develop in the brush-tailed possum by administration of FSH, suggesting a key role for this hormone in the regulation of follicular growth (McLeod, Hunter, Crawford, & Thompson, 1999). Plasma FSH concentrations fall progressively over the period of preovulatory follicle development and rise again after ovulation (Crawford et al., 1999).

For all other species, data are sketchy, with only some information on the use of LH and FSH available from studies promoting the breeding of rare and endangered marsupials. The dunnarts (Sminthopsis crassicaudata and S. macroura) have been used to establish the best methods for artificial reproductive technology for some of the rare dasyurids such as the northern quoll (Dasyurus hallucatus) and the Tasmanian devil, the latter of which currently is under significant conservation threat from a combination of facial tumor disease and land clearing (Hesterman et al., 2008a; 2008b). Luteinizing hormone and/or FSH stimulation have been used in both of these dunnart species in efforts to harvest healthy follicles for reproductive technologies (Menkhorst, Ezard, & Selwood, 2007; Czarny, Garnham, Harris, & Rodger, 2009). Similar studies have been performed on the southern hairy-nosed wombat and common wombat (Vombatus ursinus) in an effort to save the northern hairy-nosed wombat (Lasiorhinus krefftii), which is critically endangered (West et al., 2004; McDonald et al., 2006; Druery et al., 2007; West et al., 2007).

Other data are provided by the stimulation of ovarian cycles by GnRH or its agonists to promote artificial insemination of rare and endangered species, with GnRH agonists promoting normal luteal phases in the koala (Allen et al., 2008). Treatment with equine GnRH also promotes normal estrous cycling in the stripe-faced dunnart (Menkhorst et al., 2007) and the fat-tailed dunnart (S. crassicaudata) (Rodger, Breed, & Bennett, 1992), although this is dose-dependent (Rodger et al., 1992b). In the eastern grey kangaroo (Macropus giganteus), implants of potent GnRH agonists suppress reproduction, presumably because of the initial hyperstimulation of LH and FSH secretion, followed by downregulation of pituitary activities (Herbert, Trigg, & Cooper, 2006). Immunization against GnRH prevents follicular growth and development of the luteal phase in the tammar (Short et al., 1985), indicating a similar role for GnRH as that found for reproduction in eutherians.

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Sugar Gliders

Robert D. Ness DVM , Cathy A. Johnson-Delaney DVM, Diplomate ABVP (Avian, Exotic Companion Mammal) , in Ferrets, Rabbits, and Rodents (Third Edition), 2012

Marsupials: General Information

Marsupials are best known for possessing a pouch, in which the female raises its young. The degree of pouch enclosure is dependent on the species. The pouch is absent in males and in female South American short-tailed opossums, which are considered to be more primitive marsupials. The female sugar glider has a pouch containing four teats, in which she raises one or two young.

Epipubic bones (ossa marsupialia or eupubic bones) are unique to certain marsupials, but they are diminished or absent in gliders. These small bones are thought to provide an attachment for muscles that support the pouch. Their absence may be an adaptation to gliding, which reduces skeletal weight.

The metabolism of marsupials is approximately two-thirds that of placental (eutherian) mammals. The normal heart rate of a sugar glider is 200 to 300 beats per minute; the respiratory rate is 16 to 40 breaths per minute. 1 The cloaca is a common terminal opening of the rectum, urinary ducts, and genital ducts. Cloacal temperature is lower than the actual body temperature; the average cloacal temperature being 89.6°F (32°C). 1,4,8,10 True rectal temperature in marsupials can be measured by directing the thermometer dorsally into the rectum from within the cloaca. The rectal temperature is usually 97.3°F (36.3°C). 4 Measurement of the tympanic temperature is another means of determining core body temperature.

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Marsupial vocal communication: A review of vocal signal production, form, and function

Benjamin D. Charlton , in Neuroendocrine Regulation of Animal Vocalization, 2021

Didelphimorphia

The Didelphimorphia order of marsupials has one family, the Didelphidae, which are only found in North, Central, and South America (Fig. 1). Work to date suggests that adult Didelphidae have very similar vocal repertoires that consist of hisses, growls, and screeches. In contrast to some of the Australian possums (e.g., Tricosaurus sp. [21]), no mating calls have been identified. The Virginia opossum, D. virginiana, produces hisses, growls, and screeches in aggressive contexts [113]. Common opossums (Didelphis marsupialis ) and short-tailed opossums ( Monodelphis sp.) also screech and growl [29,114], and Robinson's mouse opossum (Marmosa robinsoni) produces a series of hisses when threatened [29]. The gray four-eyed possum (Philander opossum) also hisses and may utter a long chattering scream when disturbed [1]. Although technically nonvocal, it is also notable that several opossum species use the lips to produce clicking sounds during close range interactions, which are thought to act as appeasement signals and are often associated with mating [29,113–115]. In general, opossum vocal signals appear to be limited to a range of aggressive threats. It is noteworthy that southern short-tailed opossum (Monodelphis dimidiata) growls are produced on inhalation and exhalation [114], which is unusual for laryngeal vocal production, and may indicate that additional structures are used to produce relatively low frequencies in these aggressive vocalizations (e.g., the arytenoids and VVFs).

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Marsupial Neocortex

S.J. Karlen , L. Krubitzer , in Encyclopedia of Neuroscience, 2009

Visual Cortex

The organization of the primary visual area (V1) has been examined using electrophysiological recording techniques in several different marsupials, including the Virginia opossum, the white-eared opossum, the big-eared opossum, the brush-tailed possum, the Tammar wallaby, the Northern quoll, and the short-tailed opossum. As in other mammals, V1 is located at the caudal pole of the occipital lobe and contains a complete visuotopically organized representation of the contralateral visual field. The representation of the upper visual field is located caudolaterally in cortex, the lower visual field representation is located rostromedially, the horizontal meridian bisects the upper and lower visual field representations, and the vertical meridian forms the rostrolateral boundary of this field. Several studies have demonstrated that cortex immediately rostral to V1 contains neurons responsive to visual stimulation, and this area, called the second visual area (V2), has been mapped in detail in the Northern quoll. As in placental mammals, V2 contains a complete representation of the visual hemifield, with the vertical meridian represented at the caudomedial border of the field, adjacent to the vertical meridian border of V1. The horizontal meridian bisects the field and forms the rostrolateral boundary of the field. The lower visual quadrant is represented rostromedially and the upper visual quadrant is represented caudolaterally.

In studies in which electrophysiological recordings were combined with architectonic analysis, V1 has been shown to be architectonically distinct from V2, and its appearance is similar to that described for V1 in placental mammals. In Northern quolls, V2 stains lightly for both myelin and cytochrome oxidase (CO), and in favorable preparations V2 is actually composed of heterogeneous light and dark patches in CO-stained tissue.

The cortical connections of V1 are similar in all species examined. For example, in the Virginia opossum, brush-tailed possum, mouse opossum, Tammar wallaby, big-eared opossum, and short-tailed opossum, V1 is densely connected with V2 (peristriate cortex), cortex just lateral to V2 in multimodal cortex (parietal cortex and PP), the caudotemporal area (CT), posterolateral peristriate cortex, and perirhinal cortex. Furthermore, the connections of V1 are not restricted to the representation of the vertical meridian, as is the case for a number of placental mammals. Instead, connections of V1 are found throughout V2 and CT of the ipsilateral hemisphere, although the connections with V2 are patchy and appear to be related to heterogeneities identified using CO stains. Studies of the overall pattern of commissural connections also demonstrate a patchy distribution with V1 and V2 of the opposite hemisphere, indicating that V2 may be modularly organized as it is in primates and other mammals.

Thalamocortical connections of V1 in marsupials are similar to those described in a number of placental mammals. In the brush-tailed possum, the Virginia opossum, the big-eared opossum, the Tammar wallaby, and the short-tailed opossum, the primary source of input to V1 is from the dorsal division of the lateral geniculate nucleus (LGd), with moderate inputs arising from the lateral posterior nucleus (LP). In the brush-tailed possum, the primary source of input to V2 is from LP, with sparse input from LGd. In the Virginia opossum, the lateral intermediate nucleus also projects sparsely to V2.

Taken together, the data indicate that visual cortex in marsupials contains at least two cortical areas, V1 and V2. Each field is topographically organized similar to that in placental mammals. Furthermore, features of neural response properties, histochemical appearance, cortical and subcortical connections, and even the presence of a modular organization are common for visual cortex across all mammals.

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Origin and evolution of the Pantepui biota

Valentí Rull , in Biodiversity of Pantepui, 2019

Mammals

The tepuian broad-nosed bat Platyrrhinus aurarius, endemic to the Guiana Shield, and its sister species, Platyrrhinus infuscus, from western Amazonia, have their more recent common ancestor in the Andes, which suggests Quaternary or older range expansion from this cordillera (Velazco and Patterson, 2008 ). A similar pattern was found for the short-tailed opossum, Monodelphis reigi, whose Andean emigration was first proposed to have occurred during the Miocene, but no dated phylogenies were provided (Lim et al., 2010). A further phylogeographic analysis of the whole genus placed the origin of M. reigi in the Quaternary (1.2–2.4   Ma) and suggested that its more recent ancestor was an Amazonian species that eventually climbed to the tepuis, although the mechanism is not specified (Pavan et al., 2016). Voss et al. (2013) described a new Pantepui endemic species of the didelphid marsupial, Marmosops pakaraimae, and found that its sister species lived in the adjacent lowlands. This finding, together with other biogeographic evidence, was used by these authors to suggest that the Pantepui endemic mammals are "…neither ancient relicts of tepui vicariance nor descendants of long-distance-dispersing Andean progenitors" but "…evolved from lowlands species in the late Cenozoic". Podoxymys roraimae is a mouse restricted to the Roraima-tepui (Leite et al., 2015). Based on its geographical distribution and its molecular phylogenetic relationships with other genera of the same tribe (Akodontini), the CCT and the HST were considered unlikely by Leite et al. (2015). The DDT was favored, but the more likely source area would have been not the Andes—as originally proposed by the DDT—but the Brazilian Shield, located to the southeast, which is topographically isolated from the Guiana Shield and of much lower elevation than Pantepui. According to Leite et al. (2015), Podomyxys diverged from its closest relative at the end of the Pliocene (2.5–3.7   Ma), but when and how this monotypic genus reached Pantepui is still very speculative. Pavan et al. (2016) compared the origin of P. roraimae with the evolutionary history of M. reigi and concluded that "…the mammalian fauna of Pantepui is descendent from ancestors with heterogeneous distributions and habitat preferences".

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The comparative genomics of tammar wallaby and Cape fur seal lactation models to examine function of milk proteins

Julie A. Sharp , ... Kevin R. Nicholas , in Milk Proteins, 2008

A role for milk in the control of mammary function in the tammar wallaby

The expression of milk protein genes is regulated concurrently by systemic endocrine factors, by paracrine factors such as the extracellular matrix and by autocrine factors secreted in the milk. Previous studies using a tammar wallaby mammary explant culture model (Nicholas and Tyndale-Biscoe, 1985) have shown that different combinations of insulin, cortisol and prolactin are required for expression of the α- and β-casein genes, and whey protein genes including α-lactalbumin and β-lactoglobulin (Simpson and Nicholas, 2002).

Tammar wallaby mammary explants from late pregnant tammar wallabies can be induced with insulin, cortisol, prolactin, thyroid hormone and oestrogen to express the WAP gene (Simpson et al., 2000). Therefore, the inhibition normally observed in vivo during Phase 2A, and the subsequent induction of WAP gene expression around 100 days post-partum, may be hormonally regulated. In addition, the LLP genes can be down-regulated in mammary explants from Phase 3 tammar wallabies and then restimulated with insulin, cortisol and prolactin, but expression of these genes cannot be induced in mammary explants from pregnant tammar wallabies with any hormone combination tested (Trott et al., 2002 ). Either the appropriate hormonal milieu was not used and additional hormones are required, or the tissue requires additional serum or local mammary factors to express these genes.

This conclusion is supported by an earlier study showing that constructs with up to 1.8   kb of the LLP-A gene promoter did not express a reporter gene after transfection into CHO cells incubated with insulin, cortisol and prolactin, whereas control experiments showed that a rat β-casein gene construct was hormone responsive. In addition, the same construct was not expressed in lactating transgenic mice (Trott et al., 2002).

Recent studies in our laboratory have demonstrated that constructs comprising short-tailed opossum LLP-A and tammar wallaby LLP-B promoters (up to 5  kb of DNA) with a reporter gene were not transcriptionally active following transfection into HC-11 cells, regardless of the hormonal combination in the culture. However, unlike the LLP gene promoters, constructs with various WAP promoter fragments from tammar wallaby, short-tailed opossum and stripe-faced dunnart showed increased transcriptional activity when prolactin was added to the medium (D. Topcic and K. R. Nicholas, unpublished data). This suggests that the mechanisms controlling the expression of these milk protein genes in marsupial species are likely to be different from those in eutherians.

There is increasing evidence to suggest that milk plays an important role in regulating mammary epithelial function and survival, and this is particularly evident during involution (Brennan et al., 2007). Apoptosis was induced preferentially in the sealed teats of lactating mice (Li et al., 1997; Marti et al., 1997), while the litter suckled successfully on the remaining teats, which indicates that cell death is stimulated by an intra-mammary mechanism that is sensitive to milk accumulation (Quarrie et al., 1995). A protein known as the feedback inhibitor of lactation (FIL) has been suggested as a candidate and is secreted in the milk of the tammar wallaby (Hendry et al. 1998) and other species. It acts specifically through interaction with the apical surface of the mammary epithelial cell to reduce secretion (Wilde et al., 1995).

More recent studies using the tammar wallaby mammary explant culture model (Nicholas and Tyndale-Biscoe, 1985) to examine the process of involution have confirmed the likely role of milk, and particularly putative autocrine factors, for controlling mammary function during involution (Brennan et al., 2007). Mammary explants from pregnant tammar wallabies were cultured for 3 days with insulin, cortisol and prolactin to induce milk protein gene expression. To mimic involution, all hormones were subsequently removed from the culture medium for 10 days to down-regulate expression of the milk protein genes (Figure 2.9). Surprisingly, the explants retained the same level of response during a subsequent challenge with lactogenic hormones.

Figure 2.9. Northern blot analysis of β-lactoglobulin, α-casein and α-lactalbumin gene expression in tammar wallaby mammary explants. Gene expression is shown in: mammary tissue from day 24 pregnant tammar wallabies prior to culture (t0); explants cultured in media with insulin, cortisol and prolactin (IFP) for 3 days; explants subsequently cultured in the absence of hormones (NH) for 10 days; and following the re-introduction of IFP for 3 days. The length of culture in days is shown by the subscript. Total RNA (10   μg, lower panel) was assayed by Northern blot analysis using [a-32P]dCTP -labelled cDNA probes for the β-lactoglobulin, α-casein and α-lactalbumin genes (upper panels). Arrows indicate transcript size in nucleotides and RNA ribosomal bands.

Previous studies have shown that there is limited secretion of milk proteins from tammar wallaby mammary explants, but it is unlikely that milk constituents accumulated to elevated concentrations (Nicholas and Tyndale-Biscoe, 1985). The maintenance of epithelial cell viability and hormone responsiveness in explants cultured in the absence of hormones is consistent with a more active mechanism, such as the accumulation of local factors in the milk being the primary stimulus for apoptosis of mammary epithelial cells in the tammar wallaby mammary gland. This model permits the uncoupling of hormone and milk-regulated involution and, clearly, a primary outcome of these studies is evidence for the extraordinary capacity for the survival and maintenance of hormone responsiveness by tammar wallaby mammary epithelial cells cultured in a chemically defined medium with no exogenous hormones and growth factors.

Assuming that bovine mammary epithelial cells show similar characteristics to tammar wallaby mammary epithelial cells, it is likely that milk components will play a major role in the shape of the lactation curve in dairy cattle following peak lactation at approximately 20 weeks of milk production. The lactation cycle in dairy cattle includes a period of increasing milk yield in early lactation followed by a steadily declining yield for the remainder of lactation. The amount of milk produced during lactation is determined by the peak yield and the persistency of lactation (see McFadden, 1997). Milk production is largely a function of the number and the activity of secretory cells in the udder, which decline significantly between the time of peak yield and late lactation. Therefore, it follows that approaches to address the decline in milk yield and lactational persistency after peak lactation must involve changes to the frequency of apoptosis in mammary secretory cells.

Endocrine treatment of cattle with bovine somatotrophin increases milk yield but, in many cases, persistency is not altered. Furthermore, although moderate heritability of persistency suggests that selection for this trait is possible, it is achieved at the cost of milk yield. More recently, increased frequency of milking has been shown to increase milk yield, suggesting that the mammary gland has a local intrinsic resistance to regression. Identification of the milk components that impact significantly on mammary cell fate may provide new approaches for strategies to improve lactational persistency.

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Phylogeny and Comparative Physiology of Mucosal Immunoglobulins

Charlotte S. Kaetzel , Michael W. Russell , in Mucosal Immunology (Fourth Edition), 2015

Monotremes and Marsupials

A marsupial Ig resembling IgA in placental mammals was first identified in the quokka (Setonix brachyuris), with both high- and low-molecular-weight forms found in serum and the high-molecular-weight form exclusively in milk (Bell et al., 1974). Local immunization of the mammary gland with Brucella abortus elicited an antibody response in milk in the absence of circulating antibodies, suggesting local production of Ig's. Subsequent advances in molecular cloning led to the identification of the heavy-chain genes encoding IgA, IgG, and IgE in two marsupial species, the short-tailed opossum ( Monodelphis domestica) and the common brushtail possum (Trichosurus vulpecula), and in two monotremes, the duck-billed platypus (Ornithorhynchus anatinus) and the short-billed echidna (Tachyglossus aculeatus) (Aveskogh and Hellman, 1998; Belov et al., 1998, 2002; Belov and Hellman, 2003; Vernersson et al., 2002). Studies on lactation in the brushtail possum identified two stages of transfer of secretory IgA (S-IgA) into milk, the first occurring briefly after the birth of the young, when they transfer to the pouch to complete their development, and a second burst of S-IgA transfer just before the young exit the pouch after completing their "external gestation" (Adamski and Demmer, 1999, 2000).

In 2009, two groups independently described the structure of the entire IGH locus in the platypus (Gambon-Deza et al., 2009; Zhao et al., 2009), based on the previously published genomic sequence for this monotreme. Eight CH genes were located in a region spanning approximately 200   kb, arranged in the order Cμ-Cδ-Co-Cγ2-Cγ1-Cα1-Cε-Cα2. The gene encoding platypus IgD lacks a hinge region and is more similar in structure to the IgD in amphibians and fish than the IgD of eutherian mammals. Downstream of the Cμ gene is a novel Co gene (omicron for Ornithorhynchus), structurally different from any of the five known mammalian Ig classes, which may have evolved from an ancestral IgY gene. The two IgG subclass genes and the one IgE are structurally and genetically homologous to their eutherian counterparts. A phylogenetic analysis of Ig heavy-chain classes in tetrapods indicated with high statistical probability that the IgA genes evolved from an IgX gene in a common ancestor of all tetrapods, whereas the IgG and IgE genes evolved from IgY (Mashoof et al., 2013) (Figure 1). The overall similarity in Ig heavy-chain genes between monotreme, marsupial, and eutherian mammals supports the theory that all the postswitch isotypes in mammals arose before the evolutionary separation of monotremes from marsupials and eutherians about 150–170   million years ago (Belov et al., 2002). In contrast to the two IgA subclasses in the platypus, only one IgA gene has been identified in another monotreme, the echidna, and in rodents, the most closely related eutherian species. The expression of two IgA subclasses in the platypus was subsequently confirmed by molecular cloning of two IgA mRNA transcripts with unique sequences (Vernersson et al., 2010). Interestingly, the presence of multiple IgA subclasses has emerged independently in a number of mammalian species (Figure 2), suggesting a convergent pattern of gene duplication at this locus.

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