The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland

Abstract

Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism occurring in most cell types. However, this housekeeping gene is expressed at very high levels in a number of mammalian tissues including the lactating mammary epithelium, suggesting additional roles for XOR in these tissues. Mice with targeted disruption of XOR were generated to assess these potential additional roles. XOR-/- mice are runted and do not live beyond 6 wk of age. Strikingly, however, XOR+/- females, although of healthy appearance and normal fertility, are unable to maintain lactation and their pups die of starvation 2 wk postpartum. Histological and whole-mount analyses showed that in XOR+/- females the mammary epithelium collapses, resulting in premature involution of the mammary gland. Electron microscopy showed that XOR is specifically required for enveloping milk fat droplets with the apical plasma membrane prior to secretion from the lactating mammary gland. We present evidence that XOR may have primarily a structural role, as a membrane-associated protein, in milk fat droplet secretion and thus XOR provides another example of "gene sharing". About 5% of women experience primary lactation insufficiency. The above observations suggest that human females suffering from xanthinuria, a deficiency in XOR, are potential candidates for lactation problems.

Figure 1

Targeting strategy for disruption of the XOR gene and pathology of pups raised by XOR+/− mothers. (A) A cassette containing pGFP/loxP/tACE-Cre/neo^r/loxP was inserted into the HindIII restriction site of the third exon of XOR genomic sequence, creating a loss-of-function mutation. Exons are shown as boxes and identified numerically. (B–E) Pups of wild-type (WT) and XOR+/− mothers at P12. Pups from XOR+/− mothers do not have the body fat stores of brown fat (C) and mammary fat pads (E) that age-matched pups from wild-type mothers have (B,D). bf, brown fat; mfp, mammary fat pad; s, scapula. (F) Average body weight increase of pups nursed by wild-type (WT) and XOR+/− mice. Pups of XOR+/− mothers start to die at lactation day 12. Each data point represents the average of 3 litters each with 6 pups (n = 18). Data are means ± S.D. Magnification: B,C, 2.5×; D,E, 4×.

Figure 2

Histological analysis of normal and XOR+/− mammary glands. (A–P) Paraffin sections of mammary glands taken at various times during lactation and stained with hematoxylin and eosin. XOR+/− mammary epithelium undergoes destruction within the first half of lactation. Glands at lactation days 1 (A,B,I,J), 5 (C,D,K,L), 11 (E,F,M,N), and 13 (G,H,O,P). WT mammary glands are shown in A,C,E,G,I,K,M,O. XOR+/− mammary glands are shown in B,D,F,H,J,L,N,P. Magnification, 125× and 500×.

Figure 3

Whole-mount analysis and XOR enzymatic activity of wild-type and XOR+/− mammary glands. (A,B) Whole-mount analysis of wild-type (WT, A) and XOR+/− (B) mammary glands at lactation day 13. The milk-secreting alveoli are collapsed in XOR+/− mammary glands compared to wild-type, while the ducts are unaffected (red arrows). Tissue was stained with indigo carmine alum. (C) Reduced XOR enzymatic activity in terminally differentiated XOR+/− mammary glands. XOR activity in mammary glands of wild-type (WT) and XOR+/− mice was measured during late pregnancy (P) and early lactation (Lac). XOR activity is expressed as micromolar of isoxantho-pterin formed/minutes per milligrams of total mammary gland protein. Each value is the mean ± S.D. of eight separate tissue extracts. Magnification: A,B, 31.25×.

Figure 4

Milk fat droplets accumulate in XOR+/− mammary epithelium and less fat is formed in XOR+/− milk. (A,B) Histological analysis of resin-embedded mammary glands of wild-type (WT, A) and XOR+/− (B) mice at lactation day 1. The red arrows indicate XOR+/− epithelial cells that try to force out accumulated fat droplets. L, lumen of the mammary alveoli. (C,D) Lactating wild-type (WT) and XOR+/− mice were milked for milk fat analysis. Light microscopic analysis of milk samples show that XOR+/− (B) milk contains fewer fat droplets than milk from wild-type (WT, A) mice. (E) Creamatocrit analysis estimates 23.9% of average milk fat in WT mice, while only 14.7% in milk of XOR+/− mice. Values are the mean ± S.D. of 10 separate milk samples from lactation day 2. Magnification: A–D, 500×

Figure 5

The mechanism of milk fat droplet enveloping with the apical plasma membrane is disturbed in XOR+/− mammary glands. (A–F) TEM analysis of wild-type (WT, A) and XOR+/− (B–F) mammary glands at lactation day 3. Defective enveloping results in several fat droplet secretion phenotypes in the XOR+/− mammary glands. L, lumen of the mammary gland filled with casein micelles. (G–L) TEM analysis of milk in mammary alveoli at lactation day 3 from wild-type (WT, G) and XOR+/− (H–L) mammary glands. Various deformed fat droplets and numerous cell fragments are found in milk from XOR+/− mice. Magnification: A–F, 6000×–10,000×; G–L, 5000×–15,000×.

Figure 6

Various membrane defects in milk fat droplets from XOR+/− mice. (A–D) TEM analysis of milk fat droplets from XOR+/− mammary glands at lactation day 3. (A) The white arrow points to a bilayer membrane of correctly enveloped fat droplet areas as found in wild-type. The black arrow points to a completely unenveloped droplet. The white arrowhead points to an unidentified membrane structure discussed in the text. (B) Higher-power view of the boxed area in A. (C) Three membrane phenotypes can be found on a single XOR+/− milk fat droplet. The white arrow points to a portion of normal membrane bilayer. The black arrow indicates a piece of cytoplasmic crescent. Another portion of the fat droplet is occupied by the unidentified membrane structure as seen in A and B (white arrowhead). (D) XOR+/− milk fat droplet with a portion of cytoplasmic crescent that is only loosely associated with the droplet. (E) TEM analysis of an entire alveolus in a XOR+/− mammary gland at lactation day 3. The arrows point to completely destroyed mammary epithelial cells. (F) Higher-power view of boxed area in (E). Numerous free-floating mitochondria and ER are seen in burst cells of XOR+/− mammary glands (white arrows). N, nucleus. Magnification: A, 30,000×; B, 70,000×; C,D, 50,000×; E, 1000×; F, 3000×.