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Echoes of separation

Stress generated by brief absences of the mother in the days after birth damages the brain in laboratory animals and causes infertility in the offspring

ReproductionDetail from The Virgin and the Boy, Thierry BoutsReproduction

Nobody doubts that too much stress can harm sexual performance and reproductive functions. But who would suspect that the psychological pressure of staying a mere two minutes a day distant from the mother, during the first ten days immediately birth, is so intense, to the point of causing irreversible brain damage in newly-born rats? Moreover, who would believe that these lesions to the nerves arising in the most tender age of infancy could prompt a picture of infertility in a major part of these animals during their adult life, characterized by a reduction in libido, by hormonal changes, and, in females, by a difficulty in ovulating, and, in males, of producing spermatozoids?

Researchers from São Paulo and from Rio Grande do Sul, who are working in conjunction on a thematic project supported by FAPESP, not only believed in this hypothesis, but are also now defending it publicly, in an article to be printed in the October issue of the American scientific magazine Behavioral Neuroscience. In the study, the team shows that the rodents who were momentarily separated from their mothers straight after birth show, when adults, a reduction in the order of 30% in the number of nerve cells in a small region of the brain, the locus coeruleus. Classically linked to the response to stress, this nerve structure has more recently come to be related with sexual behavior and to the workings of the reproductive system. Apparently, the cause of the reduction in the quantity of neurons in the locus coeruleus was precisely the 20 minutes that these animals were kept away from their mothers in the course of the first ten days of their lives.

In a second work, with results that are still preliminary and not yet published in scientific magazines, the researchers found a significant fall in the quantity of neurons in another structure of the brain involved in the response to stress and in the reproductive process, the paraventricular nucleus. “Stress in the neonatal period produces definitive and stable marks in a few specific areas of the central nervous system, which, in turn, leads to changes in sexual behavior and reproductive capacity during adult life”, explains physiologist Janete Anselmo-Franci, from the University of São Paulo (USP), in Ribeirão Preto, the coordinator of the project and a great scholar in the role of the locus coeruleus in the female reproductive system.

“They are not generalized alterations in all the areas of the brain, but rather damage in localized regions”, comments physiologist Aldo Lucion, from the Federal University of Rio Grande do Sul (UFRGS), who heads up the Rio Grande nucleus linked to the project. The locus coeruleus and the paraventricular nucleus are not structures that are exclusive to rodents they are also present in other mammals and in human beings.

Far from the mother
The loss of nerve cells in these areas of the brain was produced in newly-born rats that were submitted to a kind of intervention called, in the technical jargon, neonatal manipulation. For the layman, an expression like this leads, erroneously, to the idea that the offspring was literally molested or maltreated straight after being born, which would explain their high degree of stress and the permanent brain damage manifested later. But this is not what the expression means. Neonatal manipulation a form of causing stress used in experiments on rats since the 50’s is characterized by the momentary and, at first sight, almost harmless interruption of the close mother-child relation that is established in the days that follow the birth.

The researchers intervention, though, was limited to the gesture of separating the offspring from the mother for two minutes in every 24-hour period. During those 120 seconds a day they were kept away from their mothers, the rats would remain gently accommodated in the gloved hands of a researcher. This procedure was repeated between the first and the tenth day of the life of the little rats, which covers the most critical part of the neonatal period in rodents.

The effects of neonatal manipulation are almost instantaneous and persist, or are even aggravated, with time. On the eleventh day of the animals life, only 24 hours after the experiment has finished, it is already possible to see a pertinent reduction in the quantity of neurons in the two areas of the brain under study. When the rats enter the adult phase, the lesions continue there, sometimes in an even more severe form, an indication that the damage in points of the central nervous system must be everlasting. The damage is manifested both in male and female animals, although a greater magnitude is shown in female rats. Perhaps this is due to the suspicion that their reproductive system above all the process of ovulation is more dependent on the activity of the locus coeruleus, the nerve structure studied in greater detail in the project.

For having an ovarian cycle very similar to a woman’s, female rats are frequently used in studies that focus on female reproduction. Their pattern of ovulation is also spontaneous and regular with the difference that their complete cycle lasts only 4 or 5 days, while the woman’s lasts 28 days on average. “With rats, we can observe the same phenomena that happen in women, in a quicker way”, says Janete.

Rats are born with about 1,500 neurons in this structure, a nerve center in the cerebral trunk not very far from the spinal cord, which in human beings is pigmented (hence the name, which means, in Latin, blue place). When the females are submitted to the process of stress derived from neonatal manipulation, there is, as time goes by, a slight increase in the quantity of neurons in this place. They go so far as to show 1,800 nerve cells in the locus coeruleus in the adult phase, at the age of 90 days. The stressed rats, though, which were deprived of maternal contact for a few minutes at the beginning of the neonatal period only one day after the end of the manipulation showed 970 neurons in the locus coeruleus.

One third less than at the moment of birth. “It is still not known what causes the death of these nerve cells”, says Lucion. And the worst thing is that this quantity of neurons practically does not increase when the females reach the adult phase. Besides confirming the loss of neurons, the researchers measured another parameter that shows the retraction of the locus coeruleus in the females. In the adult phase, the volume of this structure in rats that had been stressed in the neonatal period was almost one quarter smaller than found in females that had not been manipulated (0.037 cubic millimeters against 0.050 cubic millimeters). In male rats, practically the same thing happens: both the number of neurons and the volume of thelocus coeruleus prove to be smaller.

The brain lesions arising from the stress in the neonatal period, which cause an imbalance in the release of sex hormones, have a direct repercussion on sexual interest and capacity for procreation. From the behavioral point of view, there is less eagerness for the reproductive act. “The males almost do not pursue the females at the time of ovulation, they mount their partners less, ejaculate little, and their production of spermatozoids is reduced”, says Lucion. The female rats, the main focus for study in the works, also act outside the standard. When they are on heat, they almost do not show any lordosis, a movement intended to stimulate the penetration by the male, characterized by the uplifting of the hindquarters and exposure of the genitalia. “The majority of the females do not ovulate, and two thirds do not manage to become pregnant”, explains Lucion, from UFRGS.

It would be dangerous to speculate, in an exercise of simple mechanical transposition, that the brain damage found in animals  and the sexual and reproductive problems arising from these lesions should also occur in human babies who, for some reason, did not receive due attention from their mothers or from someone who represents the maternal figure during the first days or months of life. Although there are many similarities between the central nervous systems in rats and in human beings, there are also differences that cannot be neglected. In works that try to map the impact of a stressful stimulus in the newly-born, like those by the team from USP-UFRGS, one detail needs to be taken into account: at the moment of birth, the brain of the little rat that has just come into the world is at a more backward stage of formation than the brain of a recently-born human. In this phase of development, their vulnerability to external aggressions, such as a stressful event, is theoretically greater than thatshownby the neurons of a baby in the neonatal period.

In spite of this, albeit dangerous, making a comparison between humans and rats is far from being bereft of logic. After all, much of what is known about the physiology of our species including here the area of reproduction, the key point of the studies dealt with here derives from observations carried our previously on mammals. That is why one of the lines of study for the researchers from São Paulo and Rio Grande do Sul is trying to measure possible negative effects of little interaction between mothers with postpartum depression and their recently-born children, a situation that may show some similarity with the neonatal manipulation experiment carried out with rats. The results of these works show that the lack of physical contact between mother and child and of other more subtle forms of interaction may be a stress factor for the child. “Even a brief exchange of glances between the mother and the baby can have this effect”, Lucion says.

An exaggeration? Not at all. For example, psychiatrist Gisele Manfro, from UFRGS, has found high levels of cortisol, a steroid hormone that works as a marker for stress, in the saliva of babies born to mothers who showed postpartum depression. In other words, there are consistent indications that the lack of maternal attention seems to have exercised a psychological pressure on the newly born. That much can be said. But to go on and claim that this stress, like what happens with rats separated from their mothers in the neonatal period, can cause lesions in certain regions of the brain and cause infertility in adult life is a far cry away. “Establishing this kind of relationship in humans is very difficult and would call for decades of studies”, Janete comments. “What we can say is that our works are strengthening the hypothesis that the difficulties in reproduction of some couples, in particular in the women, can derive from a psychic state of the present, or even of the past.”

To understand the wide implications arising from the loss of neurons in the locus coeruleus, one has to understand the role played by this small structure in the brain in the maintenance of sexual behavior and reproductive capacity in rats. With its 1,800 neurons, the locus coeruleus is the main noradrenergic nucleus in the brain. This is a term used to designate a group of neurons that, when stimulated, release noradrenaline, a substance intimately linked to concepts like fear and stress.

Acting like a hormone in the bloodstream and a neurotransmitter in the brain, where, alongside other substances, it acts as an intermediary in the exchange of chemical messages between neurons, noradrenaline is central to many studies about the role of the central nervous system in reproduction. “In females, the presence of noradrenaline is of essential importance so there can be a peak in the secretion of luteinizing hormone by the pituitary gland”, explains endocrinologist Celso Rodrigues Franci, from USP’s School of Medicine in Ribeirão Preto, another participant in the thematic project. “Without this peak, there is no ovulation.”

According to Janete, this function of the locus coeruleus, to stimulate the peak of luteinizing hormone that precedes and sets off ovulation, was demonstrated for the first time by her and José Antunes Rodrigues, also from USP in Ribeirão Preto, in 1985, in the Neuroendocrinology magazine. From then on, this effect has been confirmed by her team in several works published in the last few years in international magazines. In the Brain Research magazine, between 1997 and 2002, the Brazilians posted three articles about the role of the locus coeruleus. The mechanism for action of this noradrenergic nucleus and hence of a region of the brain on female reproductive capacity is very complex.

Ovulation only occurs if a succession of synchronized events and hormonal stimuli is kept up, to coordinate a series of actions and reactions in the organism. Since the 30s, it is known that the sex hormones produced by the ovary estrogen and progesterone inhibit the secretion of luteinizing hormone by the pituitary gland during the most part of the menstrual cycle. This is the basic principle of the oralcontraceptive pill, which started to be used internationally in the 60’s.

Later on, though, scientific literature showed that, paradoxically, in the middle of the menstrual cycle, around the fourteenth day, increases in the secretion of these hormones were absolutely necessary for sparking off the peak of luteinizing hormone and, as a consequence, for leading to ovulation. That is, it began to be clear that, according to the phase of the menstrual cycle, the presence of sex hormones can have opposite effects: now it inhibits the release of luteinizing hormone, and then it stimulates it. As is known, without a peak occurring in the production of luteinizing hormone, there is no ovulation. Even today, this contradictory duality in functions of the sex hormones in ovulation is not properly understood by the scholars.

Many researchers are trying, for example, to discover interactions between these hormones and the brain structures that can be decisive for ovulation to occur. Along this line of research, the group from Ribeirão Preto began to suspect that the locus coeruleus could be related to the brain circuit that acts on ovulation, when, years ago, they damaged this group of neurons in adult female rats and observed the effects of this procedure. “We saw that the females did not ovulate”, Janete explains. After carrying out a series of experiments, some very recent and with results not yet published, the group from USP formulated a model that tries to give an account of the order of the events that lead to ovulation.

Put schematically, the model can be summed up as follows. In the preovulatory stage of the menstrual cycle, the most common and active form of estrogen, called estradiol, is secreted by the ovaries and starts to act on the nerve cells of the locus coeruleus, inducing it to synthesize receptors for progesterone, another important sex hormone produced by the ovaries. At a second moment, the mature ovarian follicle, which contains the ovule ready to be expelled, starts secreting progesterone.

This hormone then links up with its receptors in the locus coeruleus, where it has to stimulate the secretion of noradrenaline. This neurotransmitter activates a group of neurons that produce the luteinizing hormone releasing hormone, called LHRH. The presence of the LHRH hormone, in turn, is the key signal for the pituitary gland to speed up the secretion of luteinizing hormone. And this is the one that, when it reaches its peak of releasing, finally stimulates ovulation. “Our model differs from the others because it advocates the idea that progesterone activates the locus coeruleus, and it is this structure that activates the LHRH neuron group”, Janete comments. “It used to be thought before that estrogen and progesterone acted on LHRH directly.”

Pregnancy in athletes
It is not all that of a surprise to find that one form of stress, like neonatal manipulation in rats, is capable of messing up the production of hormones and has a decisive influence on the sexual and reproductive performance. Some studies show that a good number of female athletes, submitted on a daily basis to great psychological pressure in training and competitions, in the quest for good results, show an irregular ovulatory cycle. For these sportswomen, albeit apparently healthy, getting pregnant may be a more difficult task than for the majority of the female population. “The same can happen to air hostesses who work on long intercontinental flights, who are always facing jet lag”, comments Franci, the endocrinologist.

In these cases, certain specialists believe that ovulation does not take place because of stress, which may lead to an excess of noradrenaline or of other substances that act as intermediaries in the neural circuits related to the secretion of luteinizing hormone. This is the opposite situation to that of the rats with a lesion in the locus coeruleus, which do not ovulate due to the lack of this neurotransmitter.

In a woman who plays volleyball or basketball, the psychological pressure for good results, coupled with an exhausting burden of exercises, may stimulate more than necessary and at the wrong time the locus coeruleus, which could pour noradrenaline into the brain without reserve. The result is a breakdown in ovulation, from the excess of the neurotransmitter. For the time being, this idea is still a hypothesis, not a totally confirmed fact. But it has its supporters. “Both the lack and the excess of noradrenaline alter the peak of luteinizing hormone, which is indispensable for ovulation to occur”, Janete explains. “Often, a female athlete only manages to get pregnant when she stops her sporting activity and her hormonal cycle gets back to normal.” As almost anything can be a stress factor (an injury, a disease, some kind of worry), the hurly-burly and concerns of modern life may be behind some unexplained cases of human infertility.

Hope for when the milk dries up
The mother of a newly born baby suffers from stress a car crash or death of a close relative and her milk dries up. Everyone has already heard a story like this about the impact of a negative event on breastfeeding. For being so frequent, this situation is the focus of studies that are trying to understand the mechanisms involved in interrupting the production of the mother’s milk. To do so, scientists are studying the processes that regulate the secretion of prolactin, the hormone that controls the production of milk. Women who are breastfeeding need to maintain high levels of prolactin for the production of milk not to be interrupted. In some way, stress upsets the concentrations of prolactin, which in women producing milk is usually high, and makes breastfeeding difficult.

One step towards a better understanding of this mechanism has been taken by researchers from the University of São Paulo (USP) in Ribeirão Preto, and from the Federal University of Rio Grande do Sul (UFRGS). They saw that blocking the action of another hormone – angiotensin II, better known for its action of constricting blood vessels, which has an impact on blood pressure  in a specific point of the brain of milk-producing rats who had been stressed prevents the expected fall in the production of prolactin arising from a psychological trauma. This effect, which may be beneficial for the production of milk, was achieved using an injection of an antihypertensive in the arcuate nucleus of the hypothalamus, a region of the brain that controls the secretion of prolactin.

“These results confirm, in a physiological model, the role of angiotensin II in inhibiting prolactin”, explains Gilberto Sanvitto, from the Basic Health Sciences Institute at UFRGS, which carried out the procedure in rodents. Since the 60s, there is evidence showing that chronic or acute stress produces an increase in the concentration of the angiotensin II hormone in the blood and in the brain. This increase, the researchers believe, is one of the factors that make the concentration of prolactin fall, which would lead to an inhibition of the production of milk.

In theory, then, the use of an antihypertensive, as was done experimentally with rats, would succeed in preventing the negative effect for the production of milk of the excess of angiotensin II originating from stress. “Blocking the action of angiotensin II in the brain is a proposition for getting round the lack of a mother’s milk from stress”, says Janete Anselmo-Franci, from USP in Ribeirão Preto. In spite of the promising results in animals, a procedure like this is still at the test stage, and its use is not recommended in clinical treatment for humans.

The Project
Female Reproductive System: Neuroendocrine Control and Effects of Stress (01/04858-2); Modality: Thematic Project; Coordinator: Janete Aparecida Anselmo-Franci/Ribeirão Preto School of Odontology/USP; Investment: R$ 1,056,714.56