Specific triggers for the process of mammary involution are unknown but autocrine feedback mechanisms have been proposed . The source of this mechanism is thought to be factors in milk that interact with the mammary epithelium and trigger involution after a period of milk stasis. It was therefore proposed that fur seals, which exhibit a phenotype where involution is delayed even after very long periods of milk stasis, may lack these factors in their milk, or may lack the machinery to initiate the involution response.
It has long been established that LALBA, a major milk component, plays a central role in the mammary gland as the regulatory subunit of lactose synthase . We show here that otariid (Cape fur seal) mammary glands express very low levels of LALBA mRNA during the lactation cycle compared with expression levels in other mammals, which show LALBA is one of the most highly expressed genes in the mammary gland during lactation . LALBA has also recently been implicated in the induction of apoptosis of a human colon adenocarcinoma cell line  and RAW264.7 cells . LALBA was therefore a likely candidate for further study in the involution process.
Comparison of LALBA expression levels between otariid and phocid seals showed otariid (Cape fur seal) LALBA expression levels were very low and suggested a negligible amount of transcription occurs from this promoter. The low level of otariid LALBA transcription was predicted to be due to an altered TATA box in otariid seals. All three otariid species displayed a T-G transversion within the third position of the TATA box creating an AAGAAA sequence. This substitution is predicted to lead to poor binding of the TATA binding protein and RNA polymerase which are necessary for transcriptional activity . The consequence of a G to T substitution in the third position has previously been demonstrated, showing transcriptional activity is reduced to 2% . Correction of this mutation in the Cape fur seal LALBA promoter failed to increase levels of transcription, suggesting that another/other mutations also play a role in preventing successful transcription of LALBA. Like most pseudogenes where evolutionary pressures are no longer required to prevent divergence, the otariid LALBA has likely undergone a number of promoter mutations, which are conserved in other lineages where LALBA is functional.
Analysis of LALBA transcript abundance showed one major transcript, LALBA(s) was generated for all otariid species, with LALBA(l) and LALAB(Δ) detected as minor transcripts. Interestingly, LALAB(Δ) was also detected in phocid species. Other species such as dogs, humans, sheep, rats, mice and pigs also have the same cryptic 5' splice sequence in exon 1, suggesting the presence of LALAB(Δ) in species other than seals.
Translation of otariid and phocid LALBA transcripts showed otariid LALBA mRNA failed to be translated into secreted protein. These data are consistent with earlier studies, which have failed to detect LALBA protein in milk of otariid species [26, 27]. These data suggest the predicted translation of two isoforms of truncated LALBA protein (Reich and Arnould ) are incorrect. The authors suggest this is the mechanism for lack of lactose in fur seal milk and that lack of lactose as a major milk osmole has facilitated the fur seal lactation strategy and prevented engorgement while at sea. Contrary to this, it has been postulated elsewhere that other osmoles are present in otariid milk, such as Fuc(α1–2)Gal(β1–4)Glc, which is also found in related species of the order Carnivora . These trisaccharides may provide an alternative osmotic mechanism to move water from the extracellular fluid into the milk. All these species have only small amounts or no lactose relative to oligosaccharides. Myo-inositol and free amino acids are also found at high concentrations in otariid milk  and exceptionally high concentrations of taurine are found in Pinnipeds . Therefore, it is likely myo-inositol and taurine may play a significant role as organic osmolites. There are other examples in nature where lactose is not required for milk production. In tammar wallaby (Macropus eugenii) milk, carbohydrate is low and lactose is absent throughout peak lactation , during which time other unknown factors act as the major osmole, demonstrating that lactose is not necessary for milk production in some species. The high level of milk production by fur seals lactating on-shore ultimately requires the presence of an osmole, be it trisaccharides or organic osmolites, while the absence of engorgement in fur seal mammary glands while at sea is likely the result of reduced milk production by downregulation of milk synthesis at the transcriptional level .
Evidence presented here shows that bLALBA causes apoptosis of mouse and human mammary epithelial cell lines and fur seal primary mammary cells, demonstrating that although LALBA is absent in fur seal milk, the LALBA-mediated apoptotic pathway is still intact in fur seal mammary cells. It is interesting to note that all cell types in the Cape fur seal mammary population responded to bLALBA, suggesting that the apoptotic response to LALBA is not limited to epithelial cells, but also affects other cells types such as fibroblast and myoepithelial cells. The loss of LALBA apoptotic activity by heat treatment confirms the active component is of protein origin and suggests the apoptotic effects observed do not occur when the protein is fragmented by digestion with pepsin. Previously a multimeric form of LALBA (MAL), isolated from the casein fraction of human milk and formed by low pH treatment and ion-exchange, has been shown to reduce cell viability in kidney, intestine, bladder, lung cell lines, lymphocytes and thymocytes, but healthy cells were not affected  and mammary epithelial cells were not tested. HAMLET (human α-lactalbumin made lethal to tumour cells) , which is chemically treated to resemble MAL in structure, is a complex of apo-LALBA combined with oleic acid. The oleic acid co-factor binding of HAMLET is very unstable and is easily displaced by foetal calf serum (FCS). All apoptosis-inducing experiments involving HAMLET need to be performed in the absence of FCS, unlike the experiments presented here, which show LALBA inducing apoptosis in the presence of 10% FCS, suggesting that the LALBA in the current study is different in structure to HAMLET. HAMLET has been shown to localize to the nucleus where it binds to histones, disrupts chromatin structure and leads to cell death . It has also been shown that negatively charged untreated LALBA can also bind to positively charged histones without the aid of oleic acid, causing aggregation  and suggesting that other natural forms of LALBA can induce apoptosis as observed in the current study. Indeed, untreated bLALBA has previously been found to have an apoptotic effect on cell types other than mammary cells. LALBA has previously been shown to induce apoptosis in human colon adenocarcinoma cell lines  and RAW264.7 cells  using the same bLALBA preparations from Sigma . Analysis in RAW2.64.7 cells showed cells exposed to bLALBA exhibited cell shrinkage, disruption of cellular membranes, accumulation of apoptotic bodies, DNA fragmentation, an increase in sub-G1 cells, increased Annexin V expression and activation of caspase 3 . These results are characteristic of the apoptotic process and show that bLALBA indeed induces a programmed cell death response via apoptosis. Similarly, we have also observed cell shrinkage, disruption of cellular membranes, accumulation of apoptotic bodies and DNA fragmentation in this study using mammary cells following exposure to bLALBA. We have also observed an increase in caspase 3 expression following exposure of HC11 cells to bLALBA for 5 hours (Brennan et al, manuscript in preparation), suggesting the same apoptotic pathway is activated in mammary cells as in RAW264.6 cells. In the current study we expressed phocid LALBA in HC11 cells without observing apoptosis of the host cell. Although expression of the transgene was observed to be high due to the presence of a CMV promoter, the amount of protein collected in the conditioned medium was determined to be less than that required for effective LALBA-induced apoptosis. In addition, stably transfected cells have been derived which express bLALBA which are also not detrimentally affected ; we suggest that these cell lines are also not capable of producing the high concentration of LALBA (0.2 to 1.6 mg/ml) necessary for induction of apoptosis.
Our data suggest that low levels of LALBA may not cause apoptosis in the in vivo epithelium. It could be postulated that LALBA acts only at a critical concentration and requires a specific amount of time in contact with the cell, or undergoes a conformational change during milk stasis in order to elicit its apoptotic potential. Involution only occurs following milk stasis and it has been previously demonstrated that LALBA concentrations increase in milk during mammary gland involution, while other milk proteins show decreases in concentration . LALBA may cause limited apoptosis in the in vivo epithelium during lactation, as seen by decreased milk production in cows as lactation proceeds . This gradual reduction in milk production over the lactation period has been referred to as 'gradual involution'. In fur seals milk production does not decline as in other mammals, but has been shown to increase as the pup grows larger. This is presumed to sustain the nutritional needs of a growing pup. The lack of LALBA in fur seals may not only allow these mammals to circumvent involution, but may also aid in avoiding gradual involution observed in other lactating mammals.
We suggest that exposure of mammary epithelial cells to LALBA may be a mechanism for mammary gland involution during milk stasis at weaning, a time when LALBA levels increase in the milk. To support this we have presented a molecular analysis of a relevant lactation model, the fur seal, which avoids involution in the presence of milk stasis and showed that these animals do not produce LALBA in their milk. In addition we have shown that mammary epithelial cells, when exposed to similar LALBA concentrations to those found in milk, undergo an apoptotic response.