Long in the Tooth

Striations in teeth reveal the pace of life.

A histological thin section of the author’s upper left third molar, showing repetitive striae of Retzius

Timothy G. Bromage

Perhaps you can learn a lot by looking a gift horse in the mouth. You may be able to learn its life history—its lifespan, age at sexual maturity and weaning, gestation period, together with body mass and a slew of other traits—all from its teeth. The teeth of pigs and primates certainly provide all that information. Under a microscope, teeth reveal two striking features. The first is a sequence of stripes. The cells that form tooth enamel undergo daily rhythms. For part of the day, they deposit enamel quickly, but for the other part of the day, they slow down. These changes in pace produce enamel that contains visible constrictions when the rate is fast, and varicosities when the rate is slow.  Depending on the kind of microscope used, this alternation in enamel structure is also called a cross-striation.  

The second notable feature is another kind of stripe. Every two to fourteen days, enamel deposition slows down dramatically, beyond its normal day-to-day low.  This slowing creates an even more prominent line in the tooth, known as a stria of Retzius.  Remarkably, the same number of cross-striations (the first kind of stripe) always occur between adjacent striae of Retzius—within a tooth, among different teeth from the same individual, and mostly among different individuals of the same species. This conserved number of stripes-between-stripes is termed the repeat period (sometimes also called the repeat interval). The repeat period remains fairly consistent within a species, but differs across species.

Until a few years ago, nobody knew what biological factors caused the slowing in tooth growth that created the striae of Retzius. Nor did anybody know why the repeat period— the number of cross-striations between striae of Retzius—varied across species. One intriguing observation was that the repeat period seemed to be greater for larger animals. Rats have a repeat period of just one (meaning their teeth only have daily stripes), while elephants have a repeat period of fourteen. The average human repeat period falls in between at about eight or nine.  

In 2009 my colleagues and I at New York University, along with several other anthropologists and biologists around the world, decided to explore the possible connection between repeat period and body size. Body size relates to various life history characteristics—for example, larger animals typically live longer, and reach sexual maturity at a later age. We wondered whether stripe patterns in teeth could give some insight into how quickly or slowly we grow, mature, and ultimately die.

The top part of this image of human bone lamellae is made up of “primary” lamellae that are formed during growth and development. The swirly structures in the lower half of the image are secondary remodeling structures. Bone can maintain and replace itself throughout life in humans by means of cells that remove tiny cylindrical volumes of bone, the spaces of which are filled in again by bone-forming cells that lay down new lamellar bone.

Timothy G. Bromage

The first step was to verify the relationship between repeat period and body size. Our methods were straightforward. We analyzed teeth from many different species, and also had weights of the animals who had donated their dentitions to our research. We focused on primates and gathered samples from forty-two different living and fossil species. The results were unequivocal: larger body mass correlated with higher repeat period. Repeat periods included one for pygmy marmosets, three for squirrel monkeys, six for chimpanzees, and ten for orangutans.

To make sure this finding was not a quirk of the primate order, we also examined teeth from elephants and their fossil ancestors. The relationship held true here too. While the two hundred kilogram Cyprus dwarf elephant had a repeat period of six, the seven thousand and four hundred kilogram Mastodon’s repeat period was fourteen. 

The correspondence between this enigmatic enamel repeat period and body size made us wonder about the bones in our bodies. After all, bone mass and body mass scale tightly together. Heavier bodies require heavier skeletons to support them. If teeth grow by means of the repeat period rhythm commensurate with body size, it seems reasonable that our bones should also grow at this rhythm, and perhaps even show markers of this growth similar to the striae of Retzius.

As with teeth, bones appear striped under a microscope. These stripes result from the way the collagen in bone is oriented. Zones of mixed obliquely-oriented collagen separate bands in which each collagen molecule more or less faces the same direction, across or up and down the bone. The bands are known as lamellae. Although lamellae look highly periodic, the pace at which they form had not been studied when we began our research.

In order to determine how much time each lamella requires to form, we acquired fluorescently labeled bone tissue. In rats, rhesus macaques, patas monkeys, sheep, and humans, a compound was administered on specific days during development that would emit light by fluorescence microscopy in bone produced that day. When looking at the bone, we then knew how many days had passed between the development of fluorescent lines. We could also count the number of lamella between these bright lines. Dividing the total number of days between fluorescent marker administrations by the number of lamella between markings told us how many days elapsed between lamella. 

Amazingly, the lamellar formation rate in bone precisely matched the striae of Retzius repeat period in teeth. This discovery suggested to us that there is a fundamental rhythm in our bodies that determines some aspect of organismal growth rate. The striae of Retzius were thus not an anomaly, but rather an outcome of this fundamental rhythm. 

We typically observe that within a group of animals, such as primates, smaller size relates to fast growth and life history, and visa versa for large animals.  How might we observe this in the growth rate of bone? Osteocytes are the star-shaped cells that reside inside our bones. Bones that grow quickly incorporate more tightly-packed osteocytes. We therefore considered osteocyte density as an index of bone growth rate. Using powerful 3D light microscopy techniques, we were able to count the numbers of cells within the femurs of humans, eight other primates, and three non-primate mammals. We found that osteocyte density decreased as body mass increased. This result confirmed that smaller animals’ bones grow faster, commensurate with their shorter repeat periods. While the osteocyte density findings told us that small animals grow faster, the rate of lamellar formation revealed that this growth is an outcome of some periodic phenomenon, repeating after a specific number of days that is highly characteristic of a given species. Could this rhythm be the key to the evolution of diverse life histories?

Before making such a broad claim, we needed to determine whether the multi-day— or multidien—rhythm could help us understand anything about life history characteristics besides their relationship with body size. It was easy to predict a relationship between bone growth rate and adult body mass, because body growth inherently involves bone growth. But a connection between bone growth rate and, say, number of offspring per litter seems less obvious. 

To examine the relationship between the multidien rhythm and various aspects of life history, we once again turned to primates. We compiled data on a host of traits for several species. Some of these measured size: body mass, birth weight, neonatal brain weight, and adult endocranial (braincase of the skull) volume. Others involved timing: estrous cycle length, gestation length, lactation length, interbirth interval, female age at sexual maturity, female age of first breeding and lifespan. A final characteristic of interest was metabolic rate, since growth and reproduction rely on energy from metabolism.

Close-up of striae of Retzius (long parallel arrows) and cross-striations (short vertical arrows)

Timothy G. Bromage
 

We found that all traits but one correlated with the enamel repeat period. The only exception was estrous cycle length. Aside from this departure, each of the size, timing, and metabolic metrics was higher for species with a greater repeat period. When many different variables are correlated, though, it is possible that some other factor is driving the relationship. In our case we found that the factor was body mass. When the effects of body mass were taken into account, repeat period only correlated with estrous cycle length, and did not correlate with any other trait. In other words, repeat period—an indicator of the multidien rhythm—exerts a strong effect on body mass, and in turn impacts each of the remaining life history traits. The only exception was estrous cycle length, which was directly related to repeat period with no influence of body mass.

At first blush, estrous cycle length seems like an irritating outlier, and its oddity is why many do not consider it a formal life history characteristic. Without this outlier, our interpretation would be straightforward: the multidien rhythm controls all aspects of life history through its effects on body mass. The difference between estrous cycle length and the other traits we studied, however, hinted at a possible mechanism underlying this rhythmic control.

On the underside of the brain sits the hypothalamus. This brain area produces hormones and controls a number of regulatory functions in the body, such as metabolic processes. One subdivision of the hypothalamus, the suprachiasmatic nucleus, sets our circadian rhythm. This structure seemed likely to be involved in the multidien rhythm as well.

But the hypothalamus does not directly control all of the body’s systems on its own. Using chemical signals, it delegates many of these jobs to the nearby pituitary. This pea-sized gland is divided into two parts with distinct functions. The front part, or anterior pituitary, handles growth, metabolism, and reproduction. The back part, or posterior pituitary, regulates the length of the estrous cycle, in addition to water balance, sugar and salt concentration in the blood, and uterus and kidney function. 

In light of this grouping of functions in the pituitary, the nonconformity of estrous cycle in our analyses is less surprising. Estrous cycle length was the only characteristic we studied within the posterior pituitary’s dominion. All other traits related to growth and reproduction would be managed by the anterior pituitary. Our finding that repeat period governs these life history characteristics via its effect on body mass, but governs estrous cycle independently of body mass, fits perfectly with the pituitary’s division of labor. Indeed, this pattern supports our hunch that the hypothalamus—the pituitary’s boss— directs the multidien rhythm. 

The evidence so far strongly advocates for the existence of a multidien rhythm in the body that regulates body size and life history. We call this rhythm the Havers-Halberg Oscillation (HHO), after Clopton Havers who in 1691 described what we now know are lamellae in bone and striae of Retzius in enamel, and Franz Halberg, the father of modern chronobiology. We suspected that the control mechanism for this rhythm resides in the hypothalamus. But there was still a major missing piece of the puzzle: how does the multidien rhythm express itself biologically to produce body size and life history characteristics?

To answer this question, we turned to pigs. Unlike the small mammals typically used in experimental chronobiological research, which only have a daily rhythm, pigs are a medium-sized animal whose growth and development were expected to oscillate on a multidien rhythm. Although we had no clear hypotheses for what the molecule should be, we strongly suspected there must be some sort of molecular signal in the body that cycles along the multi-day rhythm, and that plays a role in growth or reproduction. Two types of molecules seemed like reasonable candidates: metabolites, the products of metabolic processes, and ribonucleic acid (RNA), which directs protein creation and performs various regulatory functions. Both of these molecule types circulate in the blood. Nobody has ever examined them before, however, to search for traces of a multidien biological rhythm.

Our first task, then, was to demonstrate that blood samples could provide reliable information about the body’s rhythms. The twenty-four hour circadian rhythm is well-established, and levels of amino acids reach their peak in the late afternoon each day, while simple sugars peak at night in diurnal mammals. We drew blood every two hours for two days from a group of thirty-three young domestic pigs. When we analyzed the levels of more than two hundred different metabolites in the blood, we found that the majority rose and fell on a twenty-four hour cycle. Moreover, amino acid levels were highest in the late afternoon and simple sugars were highest at night, as expected. These results showed that if some metabolites also cycle on a multidien schedule, our methods should pick that up.

We next drew and analyzed blood samples from the same pigs every day for two weeks. The results did not disappoint: fifty-six different metabolites cycled according to a five-day rhythm. But not all fifty-six rose and fell together. They formed two groups, one of which reached its peak three days later than the other. The metabolites in these two groups serve different functions. The first group regulates cell proliferation, cell death, and protein creation. The second group degrades and salvages materials in the body. While the first group contributes to growth, it seems the second group responds by recycling the products of the first group’s activities for re-use a couple of days later.  

Imaging of femoral bone from 3 million-year-old “Lucy.” The image provides information about the degree of orientation of collagen fibers within bone, which in turn can tell us about the ability of the tissue to resist different kinds of mechanical stresses encountered in everyday life; green is collagen perpendicular with the plane of the image, and light blue represents collagen parallel with the plane of the image. Comparing the organization of bone tissue, belonging to the species Australopithecus afarensis, with other early human species, can help us to understand more about how bone structure and function has varied over human evolutionary time.

Timothy G. Bromage

The RNA patterns mimicked those of the metabolites. Two groups of RNA exhibited a five-day rhythm, with their peaks occurring on the same days as the two groups of metabolites. They also play similar roles: the first group of rhythmic RNA participates in cell proliferation, cell death, and protein synthesis, while the second group participates in degradation and salvage. These functions are all integral to an animal’s growth.

Our metabolite and RNA findings reveal a mechanism whereby a multidien cycle determines the pace of development. But the real clincher came when we looked at the pigs’ teeth. Every single tooth sample in which clear striae of Retzius could be seen had a repeat period of exactly five. The multidien rhythm we had inferred from looking at enamel and bone was the same rhythm we observed in the metabolites and RNA responsible for growth throughout the body.

One of the evolutionary implications of this work is the possible explanation of how humans have achieved greater body size variability than any other mammal except for the domestic dog. Humans have the largest spread of repeat period values among all recorded species, ranging from about six to twelve days. Our preliminary microanatomical studies of teeth from people of known life history does indeed suggest that modern humans evolved this multidien mechanism to generate the diversity of body sizes we see among populations distributed around the globe today. 

A knowledge of the Havers-Halberg Oscillation also has broader implications for human health. A near-weekly periodicity in heart rate and blood pressure has been demonstrated in humans, which we strongly suspect relates to the multidien rhythm. Stroke and heart attack tend to occur during specific stages of the daily biological rhythm, yet there is likely a near-weekly risk to which people are presently unaware. Knowing the rhythm for someone at risk could help clinicians to carefully time preventative medications. Additionally, the HHO operates in part by controlling rates of cell proliferation, and dysregulated cell proliferation is a hallmark of cancer. For chemotherapy—a near-weekly regimen—there will be an advantage to knowing what day of the week is best for a patient to receive treatment. 

Where do we go from here? First, it is important for us to understand how such a fundamental biological rhythm went unnoticed by mammalian chronobiologists. We suspect research emphasis on cellular and molecular biology pushed aside broader organismal life history inquiries. The former approach in the science of biological timing has relied mainly on the study of small mammals—mice, rats, and hamsters. These animals are excellent candidates for such research because scientists can manipulate their developmental biology over relatively short generation times. These small mammals, however, have only daily biological rhythms to guide development and lack HHO timing mechanisms. 

We need both molecular and organismal research strategies, and the continuum of strategies between them, to obtain a more thorough knowledge of biological rhythms and their regulation of mammalian life. To that end, we hope to begin exploring this continuum in a primate model. This will help us develop understanding of the general role that the HHO has played in mammalian evolution, as well as the specific significance that multidien timing has for human life. Equally interesting will be to examine how some mammalian groups may have generated their body size diversity using mechanisms other than the HHO. 

More broadly, the study of biological timing weaves into the science of metabolic ecology. This field aims to explain how metabolic rates regulate ecological processes. All hierarchical levels of life, from the individual to the biosphere, are involved in setting the rates at which resources are extracted from the environment and allocated to survival, growth, and reproduction. Biological rhythms generally, and the HHO specifically, then, are necessary considerations for achieving a more holistic impression of the fabric of life in its ecological context.