70 Years on: the Platinum Anniversary of the Solution to Milk Ejection by the Mammary Gland. Part 1: Keith Clifford Richardson

Seventy years ago, the final bricks were being put in place that established the milk ejection reflex, a classic neuro-endocrine pathway. At the start of suckling receptors in the nipple or teat send nervous impulses to the brain. Nervous pathways in the brain then induce the release of the hormone oxytocin from the posterior lobe of the pituitary gland. Oxytocin is then carried in the blood to the mammary gland where it causes contraction of myoepithelial cells that surround each secretory alveolus containing stored milk. The milk is thus forced into the duct system of the gland where it can be removed by the sucking infant.

My old lecture slide showing the milk ejection reflex - the pathway shown
in white

The Problem

Although evidence for that reflex had been obtained there was still confusion as to which cells in the mammary gland oxytocin was acting on. The description of myoepithelial cells (and their probably contractile nature) by a string of famous German histologists began in 1850. By the end of that century, it was established that there were two types of myoepithelial cell in the mammary gland: stellate around the secretory alveoli and spindle-shaped along the outside of the ducts. However, those working on milk ejection in the first half of the 20th Century were apparently unaware of the earlier work by the German histologists and got into what can only be described as a right mess. They confused myoepithelial cells with smooth muscle, with capillary adventitial cells and the basal cells of the mammary epithelium. By the 1940s, the general view seemed to be that the very sparsely distributed smooth muscle in the mammary gland was responsible.

In the late 1940s, two people working independently, sorted the whole matter out, because of the same serendipitous finding. Their results were in total agreement and with follow-up physiological studies by one of them, the whole story was complete.

Keith Richardson in London

Keith Clifford Richardson (1905-1997) was the first to do the work and the first to publish in 1949, although by the time of publication Jim Linzell (James Lincoln Linzell, 1921-1974) had completed the histological phase of his research (published in 1951).

In 2009 the late Isabel Forsyth (1936-2016) and Peggy Neville included a short account of how Richardson came to be interested in the mammary gland in their introduction to a volume of Journal of Mammary Gland Biology and Neoplasia. He was asked for assistance as a histologist by Sydney John Folley (1906-1970) of England’s then National Institute for Research in Dairying who was working on the hormonal control of mammary development and lactation in goats. Isabel, who worked in Folley’s department, reported that Richardson was, at first, somewhat reluctant since he found himself responsible for moving the Department of Anatomy of University College London back to its pre-war premises in Gower Street from its wartime home in Surrey. Eventually, however, he agreed. A long correspondence with Folley (they were of similar age) ranged from science to their mutual interest in modern art.

Both Richardson and Linzell were trying to stain nerves in the mammary gland. Both realised that the silver impregnation technique revealed the myoepithelial cells. Richardson argued that to show their contractile nature it would be necessary to obtain sections from both full and empty glands. This he did by perfusing goat mammary glands through the artery with fixative.

Richardson’s summary expresses perfectly what he found:

Myoepithelium covers the stromal surface of the epithelium of the alveoli, ducts and cisterns of the entire gland, and is thus much more abundant than is generally realized. Smooth muscle forms scattered inter-lobular bundles closely associated with the blood vessels. The theory that myoepithelial contraction is the principal factor con­cerned with ‘let-down’ and the ejection of milk is examined; other factors such as inter-lobular smooth muscle contraction, vascular changes, and elastic recoil of the stroma appear to play minor roles, if any, in this phenomenon. Hitherto, it has been assumed that myoepithelial cells are contractile because they bear structural resemblances to smooth muscle fibres. With the new technique structural changes have been found in the myoepithelium of contracted as compared with distended alveoli and ducts. These changes, together with the general orientation of myoepithelial cells, and the precise relationship between these cells and the folds in the secretory epithelium from contracted glands, are consistent with the assumption that myoepithelium is the contractile tissue in the mamma which responds to a neurohormonal mechanism involving oxytocin. 

Figure 12 from Richardson's paper: a small
contracted alveolus in surface view,
showing a myoepithelial cell with
nucleus n and branching processes

Figures 16 and 18. Same magnification
16: Myoepithelium on the surface of distended alveoli
18: Myoepithelium on contracted alveoli

Information on Keith Richardson is sparse. He was born on Christmas Day 1905 in Geelong, Victoria, Australia. He graduated from University of Western Australia in 1926, with an MSc following in 1927. As was often, indeed usually, the case at the time he did not have a PhD but whether he derided it as did many of his colleagues as ‘the German degree’ is not recorded. He was a lecturer in histology in UCL’s Department of Anatomy by the late 1930s. In the 1939 Register he was staying at the Grand Hotel, Westgate Street, Cardiff and was described as a histologist and university lecturer in the Department of Anatomy of UCL.

There are some recollections of Richardson by former students and colleagues.

Ray Guillery FRS (1929-2017):

Keith Richardson taught the histology classes during the first terms of the medical course. He was tightly organized throughout. Not only did he start and finish each lecture precisely on time, but every sentence was a complete and elegant structure. During this introductory course and for many years at UCL thereafter I learned from him a respect for order and precision. It was through the histology course that I was particularly stimulated to take an interest in anatomy. Looking back now this is perhaps surprising. The lectures were not as exciting as many others, nor as original, but they opened doors for a completely ignorant student who had not yet been taught the importance of recognizing originality, or creativity. I think Keith understood his audience. 

Ruth Bellairs:

Within the Anatomy department the third floor (the top floor at that time) was given over mainly to histology. At the south end the Reader in Histology, Mr Keith Richardson occupied the suite of rooms facing Foster Court. He was a cherubic-looking bachelor in his fifties who published little but whose high technical standards were legendary. He was a strict disciplinarian in his teaching and no chattering or whispering by the students was ever permitted in his practical classes. The north end of the third floor, now part of Physiology, was taken up entirely by the Histology teaching laboratory 

In an interview with Lynn Bindman (LB) for an oral history project of the Physiological Society conducted by Laurence Smaje (LS), Alex Cooper (AC) and David Miller (DM) at Lynn’s home in London on 30th March 2015, the conversation turned to Richardson:

LS: [Keith] Richardson. LB: Richardson, thank you. So he was in the Anatomy department and he gave nine o’clock in the morning histology lectures. LS: Starting exactly at nine and finishing exactly at five minutes to ten. LB: And I suppose they were dry but they were just fascinating because actually he gave the physiological function of every structure he talked about. So for the skin you knew why there were hairs and sweat glands and everything else. DM: These were large room lectures together with the medics, or together with other science...? LB: Yes. Science, medics and dental students. Well, the Richardson ones I think were in a smaller theatre, there were about 100 odd. LS: Oh dear. LB: I don’t know how... LS: Well, they were given in the anatomy theatre, I think. LB: Were they? I remember... LS: Well at least they were when I was there. LB: I remember Embryology theatre, actually, that’s my memory.LS: But he left, didn’t he, to go to the States. LB: Right. LS: Because they wouldn’t give him an electron microscope. LB: Right, how fascinating. But he was such a brilliant teacher.LS: He was, excellent. 

After mammary glands: Keith Richardson in the U.S.A.

And to the U.S.A. he did go. Shipping records show he visited the U.S.A. in 1956-57 but his move to the U.S.A. was in 1959. He left Southampton for New York on 25 February 1959 aboard the French liner S.S. Liberté (the former German ship Europa, received by France for war reparations). His U.S visa was issued in 1959. In 1960 and 1961 he can be found in St Louis as Associate Professor in Washington University Medical School’s Department of Anatomy. In passing it is interesting to note that in his 1949 paper he had, in modern parlance, rubbished the efforts of the chairman of that department, Ed Dempsey (1911-1975) in trying to distinguish myoepithelial cells from other cells in the mammary gland.

By 1962 Richardson was at the National Institute of Neurological Diseases and Blindness of NIH. 

His papers from his time in the American system involved electron microscopic studies of the nervous system, particularly the nature and storage and recapture of noradenaline (norepinephrine to U.S. readers) in the sympathetic nervous system. He collaborated and published with the Julius Axelrod of NIH who was awarded the Nobel Prize with Katz and von Euler in 1970. As remarked above, Richardson’s publications were few but of very high technical quality.

Floyd E. Bloom recalls Richardson at NIH:

I decided to see if I could learn some of the rudimentary methods of electron microscopy by becoming a guest worker in the NIH Campus laboratory of Professor Keith Richardson, head of the Section on Neurocytology. He agreed I could come over, and he walked me through the details of brain perfusion fixation, dissection for electron microscopy, orientation of the tissue blocks for embedding in epoxy plastics, and how to make glass knives from whole sheets of glass in order to do ultramicrotomy to acquire the less than 1000 Angstrom thick sections that could be placed on copper grids and examined in the electron microscope. Richardson had been the head research assistant in the neurocytology laboratories at University College London before coming to the NIH [He had been lecturer in histology]. Because he was one of the few Section heads who didn’t have a doctoral degree, he called himself “Professor Richardson” in the NIH Directory. (Julius Axelrod was another until he got his degree a few years before his Nobel Prize)…In addition to teaching me the fundamentals of electron microscopy, Richardson had been one of the first people to examine tissues of the peripheral autonomic nervous system. He had observed that immersion fixation with a mixture of osmium tetroxide and potassium dichromate produced sympathetic nerve fibers containing synaptic vesicles with dense granular cores, rather than the electron lucent synaptic vesicles that epitomized the nerve terminals at the neuro-muscular junction where acetylcholine was the recognized neurotransmitter. Then just before I left NIH for Yale, Richardson published a report in Science with Julius Axelrod and Lincoln Potter showing a second method for detecting norepinephrine by ultrastructural autoradiography of nerve terminals in the pineal exposed to 3H-norepinephrine; there the sympathetic nerves actively transported the norepinephrine into the nerve terminals, where it was stored in the synaptic vesicles. I was eager to test these approaches in the brain. 

Richardson’s later publications, possibly after retirement from NIH, were from the University of Maryland’s School of Medicine in Baltimore. I have found listed what appears to be text book for students, Illustrations of Light Microscopical Preparations from Various Tissues and Organs, published by the Department of Anatomy in Baltimore in 1976. The last publication I can found in PubMed was in 1977 when he would have been 72.

Keith Richardson must at some time after he retired return to London. He died in Camden on 15 February 1997, aged 91.

I have been unable to find a photograph of Richardson. If any reader knows of one or has further information please contact me.

Coming Next

Keith Richardson submitted his paper on 29 June 1948. A month previously Jim Linzell had  made his serendipitous finding that confirmed Richardson’s observations. In Part 2, I will look at Jim Linzell’s anatomical studies and how he then put the final brick in place by then observing living mammary myoepithelial cells in action.

Bloom FE. 2012. Floyd E Bloom. In, The History of Neuroscience in Autobiography, Volume 7, pages 1-56, edited by Larry R Squire. Oxford University Press.

Forsyth IA, Neville MC. 2009. Introduction: The Myoepithelial Cell and Milk Letdown; Entrance to the Multifunctional Role of Oxytocin. Journal of Mammary Gland Biology and Neoplasia 14, 221-222.

Guillery R. 1998. Ray Guillery. In, The History of Neuroscience in Autobiography, Volume 2, pages 132-167, edited by Larry R Squire. San Diego: Academic Press.

Richardson KC. 1949. Contractile tissue in the mammary gland, with special reference to myoepithelium in the goat. Proceedings of the Royal Society B 136, 30-45.

Does cortisol in a mother’s milk really affect an infant’s temperament?

Having written about lactation for more than 45 years, I have tried to avoid writing about the subject in this blog. However, I do get drawn back from time to time—and this is one of those times.

It has been known for years that biologically active substances, hormones for example, are present in milk. All sorts of claims have been made over the years that they may have effects in the infant. However, the evidence for many such claims and suggestions was not at all convincing and so Peggy Neville and I wrote a Commentary* for the Journal of Endocrinology in 1991 setting out the criteria for establishing an effect of a substance in milk on the suckling. In brief, these criteria—the equivalent of Koch’s famous postulates for the transmission of disease—must be satisfied:

• An effect in the offspring must be obtained in response to exposure to the substance in milk.
• The effect of the substance must be abolished by removal of the substance from milk and restored when that specific component is restored.
• The substance must be shown to be present and active in milk.
• The substance must be shown to retain its biological activity in the offspring to the point at which it is postulated to act or to be activated by partial digestion within the digestive tract.

I should make it clear that none of these criteria is easy to fulfil in practical terms. However, at the time and soon after, there were two sets of studies that fulfilled these criteria: those by the late Otakar Koldovsky and his colleagues in Arizona on epidermal growth factor (EGF) and those by the late Steve Frawley and his group in South Carolina on the developmental programming of the pituitary by an agent in milk. All these results were obtained in rats.

Work in this field has continued and claims of changes in milk hormone concentrations having short- or long-term effect in the offspring have been made.

A study^† that made the newspapers recently continued the theme that maternal ‘stress’ affects and programmes the behaviour of the offspring via a change in the concentration of glucocorticoids—the classical ‘stress’ hormones—in milk.

Rhesus Macaque
By Charles J Sharp
Used on Wikipedia

The study widely reported was on a captive colony of Rhesus Macaques. The authors attempted to distinguish between the effects of milk energy intake (the subject of earlier work from the same group) and cortisol (the major glucocorticoid of primates) concentration on the behavioural characteristics of the young. Multiple regression statistical models were used to try to find what was associated with what. In short, mothers who showed a higher concentration of cortisol in their milk had offspring of a more nervous/less confident temperament, an effect particularly seen in daughters. 

Teleologically, I could see the arguments that the authors were making in relation to the temperament of the offspring produced by the different mothers. However, I am yet to be convinced that the differences in temperament that were observed are a maternal effect transmitted by cortisol in milk. This is why:

In general and in all species studied thus far, milk cortisol concentrations reflect maternal plasma cortisol concentrations. I don’t want to spend time explaining the likely kinetics of cortisol movements between the maternal circulation and milk, suffice it to say that recent increases in cortisol in the mother will be seen as an increase in milk. Short-term maternal distress would therefore result in an increase in milk cortisol concentrations. I then looked at how the authors had collected the milk samples: …infants and mothers were captured in their outdoor enclosures between 7:30 and 9:00 AM and were relocated together in temporary housing. To prevent nursing, mothers were placed in mesh jackets and allowed a standardized period of milk accumulation of 3.5-4 h. This allowed infants to remain in contact with their mothers during this period. At peak lactation, the mothers and offspring were separated for the period of milk accumulation and beyond for the behavioural assessments to be made. The procedures used to obtain the milk samples—even the initial stage of capture—are ones that would result in the ‘stress’ production of cortisol. Moreover, those of us instilled with the knowledge that because the rate of milk secretion drops rapidly in ‘stressed’ mothers, great care must be taken in interpreting the quantity of milk and milk constituents produced under such circumstances. I am also not convinced that the method used to estimate milk yield and the composition of ingested milk (in terms of fat concentration) provide a sufficiently reliable measure of energy intake at times other than during the procedure to obtain the milk sample; even then no evidence is presented that stored milk removal was complete.

If the mothers are themselves variable in temperament and respond differently to the ‘stress’ of capture, wearing a mesh jacket and/or separation from their young, then might not the variation in cortisol concentrations seen reflect not the ‘resting’ cortisol concentration but the temperament of the mother in response to these distressing events. A nervous mother could produce a nervous offspring by genetic inheritance and not via a maternal effect of cortisol concentration in milk.

Looking at the data on milk cortisol concentrations in the paper, the variation is very great (19-444 nmol/l at 1 month and 21-622 at 3-4 months) with some mothers showing major changes upwards or downwards between the two samplings; in other words, the sort of pattern that could well be expected from variable maternal responses to the procedure used to get the milk samples.

In conclusion then, I do not consider the evidence sufficient at present to conclude that cortisol in milk affects the temperament of the offspring in Rhesus Macaques nor that milk intake does not. This is not to say that further experimental evidence will not support the proposed maternal effect in the future. However, this research really does illustrate the great practical difficulties in testing hypotheses on putative milk-borne signals to the offspring, just as Peggy and I envisaged when we wrote our commentary nearly 25 years ago.

*Peaker M, Neville MC. 1991. Hormones in milk: chemical signals to the offspring. Journal of Endocrinology 131, 1-3.

†Hinde K, Skibiel AL, Foster AB, Del Rosso L, Mendoza SP, Capitanio JP. 2015. Cortisol in mother’s milk across lactation reflects maternal life history and predicts human temperament. Behavioral Ecology 26, 269-281.

‘Stress’: I have used the word ‘stress’ in inverted commas because the word still means different things to different people—a point to which I shall return at some time in the future.