Can You Hear Your Biological Clock Ticking?

By: Morgan Ivens-Duran

            In the centuries preceding the Industrial Revolution, teenage pregnancy was something to be celebrated, and the more children a woman gave birth to during her lifetime, the better. Of course, at that time life expectancy was a great deal shorter than it is today, and the risk of infant mortality was around 25%.

            In our modern era, as women have begun to seek higher education and employment outside of the “traditional” housewife model, it is perhaps unsurprising that women have begun to delay childbearing. It can seem easier to put having a family on hold than finishing school, finding secure employment, or getting that next promotion. After all, magazines and talk shows are full of stories of women in their late 30’s or even 40’s who successfully have children. With all of our medical advancements, haven’t we figured out a way to beat the biological clock?

Halle Berry (41) on the cover of InStyle, February 2008. Image from  http://tinyurl.com/asuepam

            Unfortunately, waiting that long before having children isn’t always an option. Fertility begins to decline well before the onset of menopause (after which women cease monthly menstruation), and many physicians consider 35 to be the tipping point, after which pregnancy is both dangerous to the mother and unlikely to be successful. After 45, the probability of conception through natural methods, sometimes called “spontaneous conception”, is so low that most pregnancies are only possible with a combination of medically assisted fertilization and the use of either eggs frozen at a younger age or eggs from a donor in her 20’s – 30’s.

            The real-life implications of a woman’s biological clock got an unusual airing in the public forum on an episode of Fox’s “New Girl” just after Thanksgiving. Check out the clip below:

Embedded video not working? Try clicking here. 

            While Jess’s (played by Zoe Deschanel) ignorance may seem comedic, she’s not alone. Studies in Sweden, Israel, Canada, and the US have shown that most while most people understand that fertility decreases as women get older, they often underestimate the influence of maternal age on successful pregnancies, overestimate the rate of spontaneous conception, and overestimate the success of fertility treatments such as IVF (in vitro fertilization) to promote conception. Many women assume that because medical advances and better nutrition have extended life expectancy beyond the 27 years one could expect in Europe during the late 18^th century, the age at which fertility begins to decline has moved upwards as well. But while genetic manipulations to combat the natural decline in fertility are a possibility, at the moment they are more science fiction than scientific fact.           

            This begs the question of what causes this decline in female fertility, and why we don’t see the same magnitude of a decline in men of a similar age. Current research points to increasing aneuploidy, the failure of chromosomes to separate properly during meiosis, as the culprit. Aneuploidy most often results in spontaneous abortions, in which the fetus dies prior to completing development, and those babies that do survive are often born with severe developmental disabilities. Meiosis is the process of cellular division that produces eggs or sperm, depending on the sex of the individual in question. In males, once an individual has reached puberty he continually produces new sperm via meiosis. In females, the process of meiosis begins as the fetus develops in her mother’s womb, and then is put on hold. After the onset of puberty, each month one of the immature eggs, or oocytes, completes meiosis. If it is not fertilized, the egg is discarded through menstruation and the following month, another oocyte completes meiosis.

The phases of meiosis in males and females. Image from http://tinyurl.com/aoafpcb

            The lengthy period of time an oocyte spends in the interval between the initiation and completion of meiosis in females (also known as “prophase arrest”), is likely a major factor underlying the high rate of aneuploidy in older women, but the exact mechanism is not yet fully understood and likely involves multiple factors and processes. Some studies have shown that as maternal age increases, the integrity of proteins known as “cohesins”, which are crucial to proper chromosome separation, decreases. Work on other organisms that exhibit similar declines in female fertility suggests that hormones such as TGF-b and IGF, which are also involved in regulating physical growth, may also play an important role.

            But while the exact mechanisms underlying decreased female fertility with aging are of great interest to scientists and medical professionals, the average woman simply wants to know what she should do about it. When should she start trying to have kids? What medical options are available if she has trouble conceiving?

        While by no means an in-depth look at the huge variety of maternal age-related fertility treatments available today, there are essentially two main precursors: use of a donor egg or use of eggs frozen earlier in life (ie with a younger maternal age than the biological mother’s age now). Modern methods of egg freezing, also known as vitrification, address many of the issues associated with older methods of egg freezing, such as ice crystallization that renders the oocyte non-viable. Vitrification seems to represent a promising avenue for women to set aside “younger” eggs and thus preserve their fertility. However, even though vitrification is no longer considered an “experimental” treatment, there has been a great deal of push back from the medical community, insisting that vitrification should not be available to women without immediate medical concerns that threaten their fertility, and many insurance companies still refuse to cover the cost of elective egg freezing.

            As the ethical debates over techniques such as vitrification play out, I hope that we as a society will come down on the side of hopeful parents who are struggling to cope with a biological clock who’s timing was fixed millennia ago, when living to 100 was pie in the sky rather than reality. And I hope that we continue to educate ourselves about the science of infertility and the risk of involuntary childlessness, whether by combing scientific literature, talking with your doctor, or watching surprisingly educational television episodes.

References

Bretherick, K.L., N. Fairbrother, L. Avila, S.H.A. Harbord, W.P. Robinson. 2010. Fertility and aging: do reproductive-aged Canadian women know what they need to know? Fertility and Sterility 93(7):2162 – 2168.

Chiang, T., F.E. Duncan, K. Schindler, R.M. Schultz, M.A. Lampson. 2012. Evidence that Weakened Centromere Cohesion is a Leading Cause of Age-Related Aneuploidy in Oocytes. Current Biology 20:1522-1528.

Hasholoni-Delev, Y., A. Kaplan, S. Shkedi-Rafid. 2011. The fertility myth: Israeli students’ knowledge regarding age-related fertility decline and late pregnancies in an era of assisted reproduction technology. Human Reproduction 26(11): 3045-3053.

Homburg, R., F. van der Veen, S.J. Silber. 2009. Oocyte vitrification – Women’s emancipation set in stone. Fertility and Sterility 91(4): 1319 – 1320.

Hunt, P. and T. Hassold. 2008. Female Meiosis: Coming Unglued with Age. Current Biology 20(17): R669 – R702.

Knapp, Vincent J. 1998. Infant Mortality and Malnutrition in Preindustrial Europe: A Contemporary Explanation. Nutrition and Health 12(89):89 – 95.

Lampic, C., A.S. Svanberg, P. Karlström, T. Tydén. 2006. Fertility awareness, intentions concerning childbearing, and attitudes towards parenthood among female and male academics. Human Reproduction 21(2):558 – 564.

Mac Dougall, K., Y. Beyene, R.D. Nachtigall. 2013. Age shock: misperceptions of the impact of age on fertility before and after IVF in women who conceived after age 40. Human Reproduction 28(2):350 – 356.

NPR. 2012. Egg Freezing Moves Out of Experimental Realm. http://www.npr.org/2012/10/24/163558294/egg-freezing-moves-out-of-experimental-realm

Tatar, M. 2010. Reproductive aging in invertebrate genetic models. Annals of the New York Academy of Sciences 1204:149 – 155.

Parental Care in Crocodilians: Nothing “Cold-Blooded” About it!

By Michael DeLea

What adjectives come to mind when you hear the word crocodilian? Ancient? Monster? Man-eater? Perhaps you think of one of the 23 species that inhabit the world today, two of which can be found here in the United States. How about attentive parents? Crocodilians exhibit complex social behaviors, none perhaps more interesting than those demonstrated during reproduction. Not only do members of the order Crocodylia outclass the remainder of their reptilian relatives, they rank among nature’s elite when it comes to caring for their young.

Hungry Crocodile? Or attentive parent?

As observed in many reptiles, nest construction can be a complicated and time-consuming endeavor. For example, it has been documented that female American Alligators (Alligator mississippiensis) will make frequent trips to the nest site adding materials and reshaping the nest prior to egg deposition. The design of the nest plays an even more important role when you consider the fact that sex determination is a function of temperature in at least half of the extant species of crocodilians.

Juvenile Alligator mississippiensis I encountered in Southwestern Florida

During the incubation period, crocodilians have been observed demonstrating various degrees of nest attendance and defense. This behavior is in stark contrast to that of other noncrocodilian reptiles that typically lay their eggs and leave the unborn young to fend for themselves. As to be expected, it has been shown that increased attendance of a parent leads to an increased nest success rate. In addition to maintenance, the presence of a watchful parent is a very powerful predator deterrent. In a study of American Alligator nest attendance, the defensive behaviors of the alligators were broken down into a 10-step response sequence that increased in aggressiveness, depending on the perceived threat of the intruder. Protective parents therefore give their offspring a greater chance of survival by fending off predators such as raccoons, bears, and even humans.

Female American Alligator in a typical defensive posture, preparing to defend her nest from intruders.

            After what can amount to months of incubation, the eggs will begin to hatch. Even before the newborns emerge from their shells, they begin calling out to an adult. Upon hearing the calls emanating from the nest, the adult will approach and begin excavating very carefully. The babies will continue to call out to alert the adult to their location until they are unearthed. The task of the parent is not completed once the young are freed from the nest, however. To avoid having the nest flood due to natural conditions that affect water levels, there is usually an expanse of land to be traversed before the newborns can safely reach the nearest body of water. At this point, the parent will pick up their young with their powerful jaws and transport them to the water that will serve as the nursery.

Clutched between jaws that generate the largest bite force ever recorded for living animals (3,700 psi), this crocodile transports her young to safety.

            The extent to which juvenile crocodilians will be cared for varies between species. In some cases the parental care will cease shortly after transportation to the nursery, when they are left to fend for themselves. In other cases, such as the care demonstrated by the spectacled caiman (Caiman crocodilus crocodilus), females occupying nearby habitats have been known to take turns watching over each other’s offspring.

            The ancestors of today’s modern crocodilians have been traced back over 65 million years ago. During that time they have adapted well to life on earth, outlasting the dinosaurs and many other species. When you look at certain aspects of their behavior, paternal care for example, it is obvious that these creatures have evolved to be successful in an ever-changing environment and will continue to do so.

References:

Deitz, D.C. and T.C. Hines. 1980. Alligator nesting in North-Central Florida. Copeia, 2: 348-258.

Hunt, R.H. 1987. Nest excavation and neonate transport in wild Alligator mississippiensis. Journal of Herpetology 21(4): 348-350.

Kushlan, J.A. and M.S. Kushlan. 1980. Function of nest attendance in the American Alligator. Herpetologica 36(1): 27-32.

Somaweera, R. and R. Shine. 2012. Australian Freshwater Crocodiles (Crocodylus johnstoni) transport their hatchlings to the water. Journal of Herpetology 46(3): 407-411.

High on Life

A look into the neurophysiology behind the runner's high

By Leanne Fogg

Running 13 miles for fun is often thought of as crazy, but for most long distance athletes it is simply another workout that comes with a blissful reward: the runner’s high. Toward the end of a long run, I find myself easily holding a fast pace. My body maintains a smooth rhythm, executing perfect biomechanics as I float along the trail. When in company, I can share this cadence and continue for miles in silence, enjoying the sounds of our synchronized steps. I can find myself in a state of mindlessness, escaping the weight of the stressors of life. This state of tranquility and harmony is referred to as a “runner’s high,” and results after intense exercise.

Reported effects of the runner’s high mirrored the marijuana-induced state many are familiar with. Yes, runners are claiming they can get baked without smoking.

A wave of skepticism triggered the investigation of the runner’s high hypothesis – can exercise alter the neurophysiology of an athlete, leading to pain-relieving and calming effects?

Before we dive into the exercise-induced high investigation, it is helpful to understand the neurophysiological effects marijuana has on the body.

The effects of marijuana are mainly attributed to Δ⁹-tetrahydrocannabinol (THC) – the primary psychoactive component in the cannabis plant. THC alters the cell-cell communication in the nervous system. A signaling presynaptic neuron will release neurotransmitters, which will bind to their corresponding receptors on the surface of a target postsynaptic neuron. Receptors contain highly specific binding domains that only particular molecules can fit into and elicit the downstream response. Although receptors are designed for specificity, exogenous molecules, like THC, can hijack systems and trigger alterations in many biological processes.

Scientist first discovered these receptors as being sensitive to THC, and appropriately named them the cannabinoid receptors. Cannabinoid receptors come in two flavors: type 1 (CB1R) and type 2 (CB2R). CB1 receptors are primarily located in the central nervous system, as it is one of the most prevalent G protein-coupled receptors in the brain. CB2 receptors are found in tissues throughout the body, and are involved in mediating the immune response.

 PET imaging with [¹¹C]MePPEP (an inverse agonist of CB1 receptors), showing the distribution of CB1 receptors throughout the brain (Terry, 2009).

The cannabinoid receptors bind endocannabioids (endogenous neurotransmitters) including arachidonoylethanolamine (anandamide or AEA) and γ-aminobutyric acid (2-AE). Anandamide, meaning “blissful amide,” is the main agonist (effector molecule) of CB1 receptors, leading to alterations in appetite, mood, and pain sensation.

Although receptors are highly specific, CB1 receptor activation can result through the binding of an endocannabinoid or THC. CB1 receptor binding occurs through the recognition of a hydrocarbon chain motif that AEA, 2-AE, and THC all share. 

CB1 receptor agonists (highlighted: binding motifs)

Endocannabinoid receptors are a type of G protein – they are protein receptors that span the membrane of a cell and trigger a signal transduction pathway upon binding of a specific molecule. CB1 receptors are located at the axon terminals of a presynaptic neuron (the “end” of a signaling neuron). Binding to the CB1 receptor will prohibit the release of neurotransmitters, inhibiting the signal from being passed to the postsynaptic neuron (the target neuron will not receive the signal).  This process naturally occurs in our bodies and is known as depolarization-induced-suppression inhibition (DSI).

Depolarization-induced-suppression inhibition (DSI) induced through binding of CB1 receptor

Whoa there... DSI? Let’s break this down:

Depolarization-induced: This effect is triggered upon depolarization of a neuron (when an action potential arrives, the cell depolarizes, becoming more positively charged on the inside).

Suppression: CB1 receptors are located on GABAergic neurons (neurons that release the neurotransmitter γ-aminobutyric acid, or GABA). GABA is an inhibitory neurotransmitter, suppressing the target cell from depolarization.

Inhibition: GABA neurotransmitter release is inhibited through the activation of CB1 receptors.

All together now: When postsynaptic neurons become depolarized, endocannibinoids are released and travel retrograde to CB1 receptors on a GABAergic neuron. Activation of the CB1 receptor then leads to the inhibition of GABA release.

CB1 receptor activation leads to the inhibition of adenylyl cyclase (the enzyme that synthesizes cAMP), and subsequent decrease in calcium concentrations. Low amounts of calcium prevent GABA-containing vesicles from fusing out of the cell. DSI alters neuronal communication, changing the way we perceive pain and stress. 

So how is this all connected to the runner’s high hypothesis? It has been demonstrated that endocannabinoid system (ECS) activation can be induced upon exercise in humans.

 Circulating AEA levels in human subjects were significantly higher only after intense running or cycling (Sparling, 2003).

So AEA increases upon exercise, but how do we know this is a selected benefit for running animals?

A comparison between cursorial (an animal adapted for running) and non-cursorial mammals revealed elevated AEA levels after exercise in dogs and humans while no significant increase in ferrets.

A) Plasma AEA levels before (white) and after (black) high intensity running; B) plasma AEA levels before (white) and after (black) walking, (Raichlen, 2011).

This study sheds light on possible evolutionary benefits of ECS activation. Allowing animals that have pushed themselves to their limit to enter a euphoric state might allow them to better survive - maybe running even farther than they anticipated to catch prey, or even running from a predator. ECS activation could be a result of a mechanism that promotes endurance in running mammals. 

Circulating AEA has been demonstrated to correlate with the subsequent binding to CB1 receptors in specific regions throughout the brain, including the frontolimbic brain areas associated with mood. Euphoric levels reported by subjects correlated with CB1 receptor binding in the brain. 

Activation of ESC has also provided evidence of reducing pain sensitivity and sedation, further supporting the runner's high hypothesis. 

fMRI scans before and after exercise show a reduction in pain processing in the brain (Scheef, 2012).

Exercise increases AEA levels, which in turn act on CB1 receptors, leading to DSI. It is through this mechanism that we can enter euphoric states - whether that be smoking dope or going for a long run on the central coast... I prefer the latter. 

Stay tuned for more on the endocannabinoid system and the promising future in the treatment of breast cancer, depression, and obesity.

References:

Alger, B.E., Endocannabinoid at the synapse a decade after the dies mirabilis (29 March 2001): what we still do not know, The Journal of Physiology 590(10):2203-2212.

Boecker, H., T. Sprenger, M.E. Spilker, G. Henriksen, M. Koppenhoefer, K.J. Wagner, M. Valet, A. Berthele, T.R. Tolle. 2008. The runner's high: opioidergic mechanism in the human brain, Cerebral Cortex 18:2523-2531.

Raichlen, D. A., A.D. Foster, G.L. Gerdeman, A. Seiller, A. Giuffrida. 2011. Wired to run: exercise-induced endocannabinoid signaling in humans and cursorial mammals with implications for the 'runner's high, The Journal of Experimental Biology 215:1331-1336.

Scheef, L., J. Jankowski, M. Daamen, G. Weyer, M. Klingenberg, J. Renner, S. Mueckter, B. Schurmann, F. Musshoff, M. Wagner, H.H. Schild, A. Zimmer, H. Boecker, An fMRI study on the acute effect of exercise on pain processing in trained athletes, International Association for the Study of Pain 153:1702-1714.

Sparling, P.B., A. Giuffrida, D. Piomelli, L. Rosskopf, A. Dietrich. 2003. Exercise activates the endocannabinoid system, NeuroReport 14(17):2209-2211.

Terry, G.E., J. Liow, S.S. Zoghbi, J. Hirvonen, A.G. Farris, A. Lerner, J.T. Tauscher, J.M. Schaus, L. Phebus, C.C. Felder, C.L. Morse, J.S. Hong, V.W. Pike, C. Halldin, R.B. Innis, Quantitation of cannabinoid CB1 receptors in healthy human brain using positron emission tomography and an inverse agonist radioligand, Neuroimage 48(2):362-370. 

Save them with Poison

 By Mark Hamer

Something bizarre repeatable struck my eye as I spent time hiking through Southwestern Australia in December 2012. It wasn’t the massive Eucalyptus diversicolor, or the cacophony of bush bird’s singing in their canopies – though these were quite striking - but rather the ill-fitting signs warning of a pristine landscape poisoned with something called “1080”. Signs, much like the one pictured below, were boldly displayed at the entrance to almost every trailhead, park entrance, or secondary road I came across. It became clear that whatever this poison was, the Australians didn’t seem at all afraid to use it; so much so that it could be found in even the most protected of wildlife sanctuaries.

A typical sign warning that 1080 baits have been laid throughout a given area.

It turns out “1080”, better known to nobody except the most chemistry-inclined as sodium 2-fluoroacetate, is actually a fairly common poison to the continent of Australia. In fact, it is the poison’s ubiquity that lends it so useful, though we’ll get to that shortly.

Physiologically, 1080 acts as a metabolic poison and is most toxic when ingested orally by mammals. Once consumed, the toxic effects can take place over hours or even days. Sodium 2-fluoroacetate’s toxicity lies in its similarities to a key molecule in cellular energy metabolism: acetate. Under this mechanism, soluble flouracetate associates with coenzyme A to form fluoroacetyl CoA. Fluoroacetyl CoA then enters the TCA (tri-carboxylic acid) cycle where it reacts with citrate synthase to produce fluorocitrate, a metabolite of which then binds very tightly to aconitase. This effectively halts the TCA cycle, leading to catastrophic cellular starvation and eventual necrosis. Sublethal doses may result in tissue damage, especially to tissues with high energy demand. Urinary excretion usually takes place within 72 hours of ingestion.

A schematic covering the major steps of the citric acid cycle.

At this point you may be asking: why are Australians putting 1080 poison everywhere and what the hell are they thinking? To my surprise, I learned that this was not an isolated exercise. Much of Australia, Tasmania, and New Zealand utilize 1080 poisoning. As it turns out, 1080’s effectiveness against mammals makes it an amazing tool, which has been exploited to deal with the issue of rampant invasive species proliferation. Its usefulness is reinforced by the fact that, though it is inherently toxic to most non-microbial species, the LD50’s (which is defined as the lethal dose at which 50% of animals die) in other animal classes like Amphibia and Aves are relatively high. The relative insusceptibility of amphibians to 1080 is especially informative, as it is usually these species that are severely affected by toxins and pollutants that leech in to the environment.  

 

Synthetically manufactured into pellets (see picture to right), sodium 2-fluoroacetate is also found naturally in approximately 35 native Australian plants. It is water soluble and degradable by many of Australia's native soil bacteria. Australian mammals, most of which are marsupials, have been allowed to evolve in the presence of this poison for millennia. This has lead to many native species with the ability to avoid, and in many cases even tolerate, an otherwise deadly poison. Meanwhile, species like red fox (Vulpes vulpes), feral cats (Felis catus), and European rabbits (Oryctolagus cuniculus) show extreme sensitivity to 1080 with LD50 of 0.12mg/kg, 0.4mg/kg, and 0.37mg/kg respectively. Species that show active hunting behaviors like the fox are thus easily targeted with baits containing the poison. Sadly, domesticated dogs are one of the most susceptible animals to this poison with a LD50 of about 0.07mg/kg.

Red fox, feral cats, and European rabbits all pose a very real threat to native Australian species as they outcompete them for food, water, and territorial resources.

The use of 1080 poison in Australia has been met with reasonable success, but there is fear that its effectiveness may not last. European rabbits have been shown in studies to adapt a tolerance to the poison. This is especially worrisome for r-selected animals like the rabbit. Tolerance in these animals could rapidly grow to the point where 1080 becomes an ineffective method of control. K-slected populations, like foxes, won't likely develop such tolerances in the foreseeable future.

Surprisingly, very little is actually known about the physiological modifications that some australian mammals have acquired to desensitize them to the effects of 1080. What is known is that the rate of de-fluorination does not play a significant role in this acquired tolerance. A study in the 1960's using fruit flies (Drosophila melanogaster) showed acquired resistance to 1080 after only 67 generations. Frustratingly, it seems there is little interest in the mechanism of mammalian resistance. It is likely, however, that modifications to certain proteins (perhaps aconitase?) involved in the TCA cycle are responsible.

References:

Gilbert S. 2012. A Small Dose of Toxicology: The Health Effects of Common Chemicals. Print.

Gooneratne, S.R., C.T. Eason, L. Milne, D.G. Arthur, C. Cook, and M. Wickstrom. 2008. Acute and Long-term Effects of Exposure to Sodium Monofluoroacetate (1080) in Sheep. Onderstepoort Journal of Veterinary Research 75(2): 127-139.

Mcilroy, J.C. 1986. The Sensitivity of Australian Animals to 1080 Poison: Comparisons Between the Major Groups of Animals, and the Potential Danger Nontarget Species Face From 1080 Poisoning Campaigns. Australian Wildlife Research 13: 39–48.

Mcilroy, J.C., D.R. King, and A.J. Oliver. 1985. The Sensitivity of Australian Animals to 1080 Poison VIII.* Amphibians and Reptiles. Australian Wildlife Research 12: 113–118.

Miller, C.J., and S. Anderson. 1992. Impacts of aerial 1080 poisoning on birds of Rangitoto Island, Hauraki Gulf, New Zealand. New Zealand Journal of Ecology 16(2): 103-107.

Proudfoot A.T., S.M. Bradberry, and J.A.Vale. 2006. Sodium fluoroacetate poisoning. Toxicology Review 25(4): 213-219.

Tahori, A.S. 1966. Changes in the resistance pattern of a fluoroacetate-resistant fly strain. Journal of Economic Entomology 59(2): 462-464

Twigg L., Lowe T., and M. Gary. 2009. 1080–Characteristics and use. Western Australian Agriculture Authority. ISSN: 1833-7236.

Twigg, L. E., G.R. Martin, and T.J. Lowe. 2002. Evidence of pesticide resistance in medium-sized mammalian pests: a case study with 1080 poison and Australian rabbits. Journal of Applied Ecology 39: 549–560.

Additional References:

http://www.daff.qld.gov.au/documents/Biosecurity_EnvironmentalPests/IPA-Wild-Dog-Fact-Sheet-1080.pdf

http://www.inchem.org/documents/pims/chemical/pim494.htm