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| This photomicrograph (from here) shows the cross section of a female mouse. The medulla (M), X-zone (XZ) and zona fasciculata (ZF) of the cortex are labelled. The outer, zona glomerulosa, of the cortex can be seen but is not labelled |
Introduction
For a few months in 1965-66 I worked on the aptly-named X-zone of the mouse adrenal in Hong Kong. In 2018 I wondered what had happened to research on the X-zone. Had its function been discovered? The answers were in fact ‘rather little’ and ‘no’ and so I offered to give a talk at an annual Society for Endocrinology meeting in an attempt to stir up some interest in a problem that has intrigued those interested in the workings of the adrenal gland since the 1920s. This I did in November 2019 at Brighton meeting of the British Endocrine Societies.
I have written below an account of the cellular origins of the X-zone because it now transpires that we were misled in the 1960s by the misinterpretation or misreporting of early findings and that, in fact, the early workers were right about where it came from during development of the embryo. In addition, nobody it seems had spotted an important paper published in 1942 that made our experiments in Hong Kong unnecessary. But first the personal historical background:
Four weeks after we had arrived in Hong Kong, I received an aerogramme from John Phillips he had written on 28 November 1965. He was on his first ‘long leave’ from the University of Sheffield and was spending it back in Sheffield with Ian Chester Jones, where, until December 1962 he had been a lecturer. We had been up to Sheffield several times before leaving for Hong Kong on 1 November and the general idea was that I should have a look to see if steroid hormones found in vertebrates occur in invertebrates as well. I was beginning to see what we had in terms of chromatographic equipment and chemicals in order to make a start when that aerogramme arrived. He wrote:
…I listened to Prof Paul Delost give a lecture on the X Zone last night and I was surprised to hear that he considers the X zone to be under medullary control. He bases this conclusion on the absence of an X zone in an adrenal in which the medullary tissue has been aspirated from the centre of the gland with a needle and vacuum pump—the other gland remaining as a control. The interesting thing about this preparation is that if you castrate the post-pubertal male the X zone reappears in the gland with a medulla but not in the other adrenal without a medulla…But the main criticism of Delost’s approach is that he destroys the vascular bed of the adrenal. This can be overcome by an operation called “enucleation” in which the whole of the adrenal is expressed leaving only the capsule from which a new adrenal cortex regenerates. Will you get some male mice and enucleate adrenals before puberty…
That I did and the results were clear. Given though the techniques available at the time it was difficult to envisage taking the approach further. The caravan moved on.
Cellular Origins of the X-Zone
After its description1 but misidentification in 1924 by Kiyoshi Masui and Yasushige Tamura of the Imperial University of Tokyo (with a further publication in English in 19262) and its naming in 1927 to reflect its unknown function by Evelyn Howard (1904-1999) then at Stanford3, the X-zone of the mouse adrenal excited the interest of pioneering endocrinologists who did not yet describe themselves as such. The structure, which develops after birth between the cortex proper and the medulla, still befits its name; the function of the X-zone remains unknown4,5. Early work was concerned with its origins and with its hormonal control since it disappears at puberty in males and during first pregnancy in females6.
In terms of hormones in the circulation, androgens cause the X zone’s disappearance while LH (luteinising hormone) from the pituitary is necessary for its maintenance4,7. Although excellent research was done in the early years on the possible cellular origins of the X-zone it is only more recently that cell lineage studies using molecular markers have provided further evidence that it is derived from the fetal or inner adrenal cortex, as opposed to the definitive or outer cortex which forms the well-known zones of the adrenal: glomerulosa, fasciculata, reticularis (references in4). Therefore, the X-zone of the mouse appears to be homologous with the human fetal adrenal cortex, which, as its names implies is present only in the developing fetus. Such an origin was suspected by some early workers who referred to the ‘human fetal X-zone’ but strongly denied by others. Other possible homologues are the juxtamedullary zones of various size and appearance observed in some other eutherian mammals (cat, rabbit, voles, hamsters, and shrews)6,8.
In this short article I first consider whether the more modern findings on the origins of the X-zone are consistent with the early studies since the over-riding impression created in the mind by reading reviews and papers from the latter half of the 20th century is that the X-zone is derived from the inner cells of the zona fasciculata or, in other words, is just another zone of the definitive cortex. Then I review the evidence from little-known perturbative experiments, published, to modern eyes, in obscure places, that throw light on the origins of the X-zone.
That the X-zone is derived from the fetal or inner cortex is entirely consistent with the findings of Harry Waring9 who was then working in Liverpool for an M.Sc. The topic was suggested to him by a forgotten promoter of endocrinology in Britain, a famed lecturer in zoology, Ruth Culshaw Bamber (1889-1970) who was always known as Mrs Bisbee. Horace ‘Harry’ Waring (1910-1980) showed in 1935 that during embryonic development there is—initially—an intermingling of the cortical and medullary elements. Remodelling then concentrates the medulla until there is a clear separation from the cortex. He identified cells, comprising what he called the interlocking zone, between the medulla and cortical elements. These cells became concentrated around the time of birth into a layer around four cells thick. The outer cortex in the meantime was growing and forming the usual zones. But it was that layer of interlocking cells that went on to form the X-zone after birth. Later, as the X-zone degenerated there was left a ‘medullary connective tissue capsule’ or ‘juxtamedullary capsule’ around the medulla formed, it was presumed, from the collapsed stroma9.
In 1928, Ruth Deanesly (1901-1997) in London had already observed that after degeneration of the X-zone some of its cells remained around the juxtamedullary capsule10. Therefore, the key early finding that a secondary X-zone forms after castration of male mice can be explained by growth from these cells, i.e. remnants, capable of division, of an inner (fetal) cortex rather than from a differentiation of cells from the inner zone (z. fasciculata in the mouse) of the outer (definitive) cortex.
I cannot explain why the view prevailed, despite evidence to the contrary, that the X-zone was part of and derived from the outer or definitive cortex. As one example, the following is from the highly influential review written by Helen Wendler Deane (1917-1966) published in 19628, four years before her early death:
These [X-zone] cells differentiate postnatally, at about 2 weeks, from the inner portion of the fasciculata (Whitehead 1933a, Waring 1935).
The problem with this statement is that neither Raymond Whitehead11, working in Manchester, nor Harry Waring9 drew any such conclusion. Only Waring of the two studied the origins of the X-zone and his conclusion was, as I have noted above, entirely different.
All the above evidence, even the sophisticated and relatively recent cell lineage studies, have been observational. Those seeking direct, experimental evidence that throws light on the origin of the X-zone would at first sight be discouraged since it all appeared over 50 years ago in obscure publications and/or written in French while one important paper had, I discovered recently, been overlooked entirely.
Until I found that paper, the first experimental work that has a bearing on the problem was thought to have been that in the 1960s from Paul Delost’s laboratory at the University of Clermont-Ferrand in France and in particular that of his student Parviz Chirvan-Nia who later returned to Tehran University of Medical Sciences in Iran. They took advantage of the fact that a secondary X-zone develops after castration in male mice. Their most pertinent finding12,13 was obtained in mice from which the adrenal medulla had been removed by aspiration through a very fine pipette, a technically difficult procedure. In males in which the entire medulla had been removed, a secondary X-zone failed to develop after castration. By contrast, if even a small piece of medulla remained, an X-zone developed around it. The effect was local; the untreated contralateral adrenal was unaffected.
All of Delost’s work from the 1950s onwards was published in French and had received little attention in the English-speaking world. However, on 27 November 1965 Delost was invited by Ian Chester Jones (1916-1996) to give a seminar in Sheffield, having been one of Chirvan-Nia’s external examiners earlier that year. His old student, John Guest Phillips (1933-1987; FRS 1981), on leave from the University of Hong Kong, was also there and while intrigued by Delost and Chirvan-Nia’s work he and Chester Jones were concerned that, in aspirating the medulla, the venous drainage from the adrenal would have been destroyed. They thought that a complementary approach, that of enucleation, in which end of the adrenal is snipped off and the medulla and most of the cortex squeezed out, would be a useful test. After enucleation the outer cortex redevelops and shows a normal zonation but without a medulla. As a result, John Phillips sent me an aerogramme the next day asking if I, having arrived in Hong Kong four weeks earlier as a PhD student, would take it on. This I did and the results were identical to those obtained by Chirvan-Nia and Delost: a secondary X-zone failed to develop if the medulla had been removed completely; if even a small remnant of medulla remained after incomplete enucleation, an X-zone developed around it14.
In 2019 while preparing a talk5 for the Society of Endocrinology on what has happened to research on the X-zone I discovered our experiments had simply confirmed what had already been published. The same approach, enucleation, had yielded the same results in 1942. The paper, which appears not to have been quoted by anybody working in or reviewing the field, was by Murchie Kilburn McPhail (1907-1987) and his student H.C. Read, of Dalhousie University in Canada15. I can only assume that it was missed because it appeared in wartime, albeit in a leading journal, when scientists were otherwise occupied. However, another paper from the same authors from the same year was picked up and referred to.
Suggestions as to possible mechanisms as a result of these experimental approaches, for example what would now be termed a paracrine effect of medullary cells on the inner cells of the cortex against the direction of blood flow, can now be discarded since the removal of the medulla would by any technique result in the extirpation of the inner cortical anlagen formed in and around the juxtamedullary connective tissue capsule after degeneration of the X-zone.
In conclusion, the original observations by Harry Waring on the origins of the X-zone and the experimental evidence on the necessity of the adrenal medulla for the presence of an X-zone are entirely consonant with the X-zone being derived from the fetal or inner cortex and not the definitive or outer adrenal cortex. The recognition that there are two populations of cortical cells only one of which is responsible for the classical zonation of the adult adrenal gland explains many of the false leads followed, blind alleys entered and bold assertions made by those working in the field during the middle decades of the 20th century.
Research over the past 96 years has established the hormonal control of the adrenal X-zone and seemingly settled its cellular origins. Will we also know its function by the time of the centenary of its discovery?
1. Masui K, Yamura Y. 1924. The effects of gonadectomy on the structure of the suprarenal glands of mice, with special reference to the functional relation between this gland and the sex gland of the female (Translated). Nihon Chikusan Gakkaiho 1, 55–79.
2. Tamura Y. 1926. Structural changes in the suprarenal gland of the mouse during pregnancy. Journal of Experimental Biology 4, 81–92.
3. Howard E. 1927. A transitory zone in the adrenal cortex which shows age and sex relationships. American Journal of Anatomy 40, 251-293.
4. Huang C-C J, Kang Y. 2019 The transient cortical zone in the adrenal gland: the mystery of the adrenal X-zone. Journal of Endocrinology 241, R51–R63.
5. Peaker M. 2019. What happened to the adrenal X-zone. Endocrine Abstracts 65 SE 1.1. DOI: 10.1530/endoabs.65.SE1.1.
6. Chester Jones I. 1957. The Adrenal Cortex. Cambridge: Cambridge University Press.
7. Gannon A-L, O’Hara L, Mason JI, Jørgensen A, Frederiksen H, Milne L, Smith S, Mitchell RT, Smith LB. 2019. Androgen receptor signalling in the male adrenal facilitates X-zone regression, cell turnover and protects against adrenal degeneration during ageing. Scientific Reports 9, 10457.
8. Deane, H.W. 1962. The anatomy, chemistry, and physiology of adrenocortical tissue. In The Adrenocortical Hormones Part 1. Handbuch der Experimentellen Pharmakologie, edited by Eichler O & Farah A, subedited by Deane HW, 1-185. Berlin, Springer.
9. Waring H. 1935. The development of the adrenal gland of the mouse. Quarterly Journal of Microscopical Science 78, 329–366.
10. Deanesley R. 1928. A study of the adrenal cortex in the mouse and its relation to the gonads. Proceedings of the Royal Society B 103, 523–536.
11. Whitehead R. 1933. The involution of the transitory cortex of the mouse suprarenal. Journal of Anatomy 67, 387–392.
12. Chirvan-Nia P. 1965. Données nouvelles sar la zone X surrénalienne de las souris. Doctoral Thesis, Université de Clermont.
Brudieux R., Chirvan-Nia P., Delost P. 1966. Sur les relations directes entre la médullo-surrénale et le cortex surrénal. Journal de Physiologie, Paris 58, 213-217.
14. Peaker M, Phillips JG,. Peaker SJ. 1967. A relationship between the medulla and the X-zone of the mouse adrenal. In Proceedings, Third Asia and Oceania Congress of Endocrinology, edited by Litonjua A, vol. 2 317–321. Manila.
15. McPhail MK, Read HC. 1942. Regeneration of adrenal gland following enucleation and transplantation with special reference to X-zone. Endocrinology 31, 486–492.
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