Keywords
circadian rhythm - pregnancy - embryonic and fetal development - light
Palavras-chave
ritmo circadiano - gravidez - desenvolvimento embrionário e fetal - luz
Introduction
Light in the environment interferes with the biological function of different systems,
and circadian rhythm activities are related to light variation. The periods of sleep
and wakefulness are directly associated with the circadian rhythm, and the restriction
of nocturnal sleep during pregnancy can affect hypothalamic hormones, plasma cortisol,
and body weight.[1]
[2]
The sleep-wake cycle may be altered by working shift, and some health issues, such
as reproductive success, mating and pregnancy problems are related to working at night
or shiftwork. Alterations in the biological rhythm caused by shiftwork are intimately
linked to changes in the female hormonal cycle, and consequently in reproductive function.[2]
[3] Melatonin, an indolamine produced by the pineal gland, plays a key role in the regulation
of the circadian rhythm. This hormone is secreted during the night and its function
in mammals is to mediate signals of darkness.[4] Environmental light is the most important factor for the regulation of melatonin
synthesis, responsible for circadian rhythm and its secretion. Exposure to light at
night acutely inhibits the synthesis of melatonin; however, darkness does not stimulate
its production.[5] It should be noted that the presence of light, even of low intensity (50–300 lux)
as found in residences, can inhibit the production of melatonin in humans.
Variations in serum melatonin levels are closely related to ovulation disorders and
the function of melatonin in the female ovarian cycle is associated with steroidogenesis.[6] Melatonin resulting from the production related to the circadian rhythm is transferred
from the mother to the fetus through the placenta or maternal milk.[7] Thus, exposure to light and the consequent deregulation of the maternal circadian
rhythm can possibly cause repercussions for the fetus.
The question proposed using an experimental animal model is whether different times
of exposure to artificial light environmental during pregnancy causes changes in morphological
and histological parameters of mother and fetus. There are no evidences yet in literature
to demonstrate the effect comparing luminosity differences in the ambiance throughout
the gestational period. Therefore, the objective of the present study is to evaluate
whether different environmental lighting conditions affect the reproductive parameters
of pregnant females and the development of their offspring.
Methods
This study was conducted at the Animal House of the Universidade de Franca within
the Maternal-Infant project of the Laboratory of Health Promotion Strategies. The
study was approved by the Ethics Committee on Animal Use of the Universidade de Franca
(Protocol number 015/15). Fifteen female albino Swiss Webster mice (Mus musculus) obtained from the Animal House of the Universidade de São Paulo (USP, in the Portuguese
acronym) in Ribeirão Preto were selected for this study. They were 90 days of age
and weighed ∼ 40 g. The animals were kept under the following conditions: constant
air renewal, ambient temperature of 22 ± 2°C, and humidity of 50%. Water and ration
were available ad libitum. The females selected for this study were mated and divided into three groups: 1)
light group, consisting of five pregnant mice kept in the presence of light for 24
hours; 2) dark group, consisting of five pregnant mice kept in the dark for 24 hours;
3) light/dark group, consisting of five pregnant mice kept under a 12/12-hours light/dark
cycle, with lights on from 6 AM to 6 PM.
The animals were mated at a proportion of one male per female, always in the morning
(7 AM). Mating was confirmed by the inspection of the vaginal plug, always 2 hours
after the female-male exposure, and according to the presence of seminal fluid in
the vagina, the test was considered positive and considered day zero of pregnancy.
The mice selected for the study were nulliparous.
The animals were kept in plastic cages (2 animals/cage) for a period of 18 days. Animals
from the light/dark group (12 hours light and 12 hours dark) were maintained on a
normal day/night cycle. The light group was exposed to constant cold light in the
room for 24 hours. Animals of the dark group were kept in a completely dark room for
24 hours. For this purpose, the windows were covered with a double layer of brown
paper, as were the shelves containing the cages. A FoxLux Timer (FoxLux Ltda., Pinhais,
PR, Brazil) was used for light control in a room with light-beige colored walls measuring
∼ 11 m2, with a rail containing two fluorescent lamps and slate floor. The light intensity
on a scale of 2,000 was: center of the room (2,000:180 lux), back (2,000:56 lux),
and front (2,000:65 lux). The experiments were conducted in the center of the room.
The animals were killed following the Brazilian Good Practice Guide for Euthanasia
of Animals.[8] Female mice were killed by intraperitoneal injection of thiopental (150 mg/kg).
Their offspring were anesthetized by hypothermia (immersion in ice for 20 minute),
followed by decapitation with a sharp blade. Females were euthanized on day 18 of
pregnancy. The fetus and placenta were immersed in saline 0.9% and transferred to
absorbent paper towels for the complete removal of fluid or any type of residue before
measurements to avoid false results. The fetuses collected were weighed on a MARTE
AL500 high-precision scale (Marte Científica, São Paulo, SP, Brazil), and their length
was measured with a caliper (millimeter scale). The fetal skull, chest and lungs,
and maternal uterus and placenta were fixed in 10% paraformaldehyde for 24 hours and
transferred to alcohol 70% before embedding them in paraffin. Routine staining with
hematoxylin and eosin (H&E) was used for histological analysis.
The results were compared between groups using the analysis of variance (ANOVA) test,
followed by the Tukey test. A p-value ≤ 0.05 was considered statistically significant. The BioStart 5.0 (AnalystSoft
Inc., Walnut, Canada) program was used for statistical analysis of the data.
Results
The results have demonstrated that the difference of luminosity in the ambiance seems
to have no influence in the female reproductive parameters, however, they suggest
that it has influence on the fetus morphometric parameters. [Table 1] shows the maternal variables. No significant differences were observed between groups.
Analysis of the weight evolution of females during pregnancy showed a similar average
weight gain and final weight in the three groups, with no statistically significant
difference. However, weight gain was lower in females exposed to light for 24 hours
compared with the other two groups, but no statistic difference was observed in weight
gain. The same was observed for litter size, with no significant difference between
the three groups. However, a smaller litter was found in the group submitted to light
deprivation during the experiment (average of 9.2 ± 4.2 offspring per litter). The
number of resorptions did not differ significantly between groups. However, no resorption
was observed in the group exposed to light for 24 hours ([Table 1]). Placental and uterine weights were also similar in the groups. The same trend
was observed for uterine weight ([Table 1]).
Table 1
Characteristics of pregnant mice sample
|
Light (n = 5)
|
Dark (n = 5)
|
Light/dark (n = 5)
|
p-value
|
|
Final weight (g)
|
|
Average
|
63.0
|
65.7
|
67.1
|
0.71
|
|
SD
|
6.6
|
10.4
|
5.0
|
|
|
CV (%)
|
10.4
|
15.9
|
7.4
|
|
|
Litter size (n)
|
|
|
|
|
|
Average
|
12.0
|
9.2
|
13.6
|
0.15
|
|
SD
|
2.7
|
4.2
|
2.9
|
|
|
CV (%)
|
22.8
|
46.3
|
21.8
|
|
|
Uterine weight (g)
|
|
Average
|
1.7
|
1.5
|
1.7
|
0.57
|
|
SD
|
0.08
|
0.34
|
0.24
|
|
|
CV (%)
|
4.8
|
22.8
|
14.4
|
|
|
Resorption (n)
|
|
Mean
|
0
|
2.4
|
0.6
|
0.26
|
|
SD
|
0
|
3.9
|
0.5
|
|
|
CV (%)
|
|
98.0
|
91.2
|
|
|
Placental weight (g)
|
|
Average
|
1.6
|
1.2
|
1.6
|
0.35
|
|
SD
|
0.4
|
0.6
|
0.3
|
|
|
CV (%)
|
29.2
|
48.5
|
24.0
|
|
|
Estimated litter (n)
|
|
Average
|
12.0
|
11.6
|
14.2
|
0.20
|
|
SD
|
2.7
|
0.8
|
2.7
|
|
|
CV (%)
|
22.8
|
7.7
|
19.5
|
|
Abbreviations: CV, coefficient of variation; SD, standard deviation.
Analysis of variance followed by Tukey test, with p < 0.05 indicating statistical significance.
[Table 2] shows significant differences in the fetal variables between the groups exposed
to different environmental lighting conditions. Average fetal length ([Fig. 1]) was significantly higher in the light group compared with the dark group (p < 0.05) and light/dark group (p < 0.01). Fetal weight was also higher in the light group compared with the other
groups (p < 0.01). On the other hand, no significant difference in weight was observed between
the dark and light/dark groups. Analysis of fetal cranial measures showed better average
growth values in the light group. The anteroposterior and laterolateral lengths of
the skull differed significantly between light and light/dark groups (p < 0.01) and between dark and light/dark groups (p < 0.01), while no difference was found between light and dark groups. Average skull
weight was significantly higher in the light group compared with the dark group (p < 0.05) and the light/dark group (p < 0.01), also, there was a difference between the dark and light/dark groups (p < 0.05; [Table 2]). Similarly, the average chest variables tended to be higher in the light group
([Table 2]). The mean superoinferior diameter of the chest was significantly greater in comparison
to the light and light/dark groups (p < 0.01) and to the dark and light/dark groups (p < 0.05). The average laterolateral diameter was similar in the three groups.
Fig. 1 (A) Photograph of the morphometric evaluation of a mouse fetus of the control group.
(B) Image of the uterus of a control mouse.
Table 2
Morphometric variables of the 134 fetuses of 15 albino Swiss Webster mice (Mus musculu
s)
|
Variable
|
Light (n = 60)
|
Dark (n = 46)
|
Light/dark (n = 68)
|
|
Length (mm)
|
|
Average
|
24.5*
|
22.8
|
22.2
|
|
SD
|
1.1
|
1.9
|
4.4
|
|
CV (%)
|
4.6
|
8.4
|
20.1
|
|
Weight (g)
|
|
Average
|
1.4*
|
1.2
|
1.1
|
|
SD
|
0.08
|
0.14
|
0.33
|
|
CV (%)
|
5.6
|
11.9
|
28.3
|
|
Anteroposterior skull (mm)
|
|
Average
|
11.2
|
11.1
|
10.3**
|
|
SD
|
0.5
|
0.5
|
1.2
|
|
CV (%)
|
5.1
|
5.2
|
11.6
|
|
Laterolateral skull (mm)
|
|
Average
|
7.4
|
7.1
|
6.7**
|
|
SD
|
0.5
|
0.4
|
1.0
|
|
CV (%)
|
7.3
|
6.0
|
16.0
|
|
Skull weight (g)
|
|
Average
|
0.33¥
|
0.30ϕ
|
0.28
|
|
SD
|
0.06
|
0.3
|
0.04
|
|
CV (%)
|
18.3
|
9.8
|
16.8
|
|
Superoinferior thoracic diameter (mm)
|
|
Average
|
10.1
|
10.0
|
9.5**
|
|
SD
|
0.8
|
0.7
|
1.3
|
|
CV (%)
|
7.9
|
7.7
|
14.3
|
|
Laterolateral thoracic diameter (mm)
|
|
Average
|
8.1β
|
7.7α
|
8.5
|
|
SD
|
0.5
|
0.6
|
0.9
|
|
CV (%)
|
6.2
|
7.8
|
11.0
|
|
Thorax weight (g)
|
|
Average
|
0.47*
|
0.40
|
0.39
|
|
SD
|
0.05
|
0.04
|
0.05
|
|
CV (%)
|
11.9
|
11.9
|
14.7
|
Abbreviations: CV, coefficient of variation; mm, millimeters; SD, standard deviation.
Analysis of variance followed by Tukey test, with p < 0.05 indicating statistical significance.
*: p < 0.01 light group vs dark and light/dark groups.
**: p < 0.01 light/dark group vs light and dark groups.
¥: p < 0.05 light group vs dark group, and p < 0.01 light group vs light/dark group.
ϕ: p < 0.05 dark group vs light/dark group.
β: p < 0.01 light group vs dark group, and p < 0.05 light group vs light/dark group.
α: p < 0.01 dark group vs light/dark group.
All placentas and uteruses were submitted to histological analysis. Placental evaluation
revealed the same normal structural pattern in all groups, showing cellular features
typical of the species ([Fig. 2]). Evaluation of the uteruses showed a discrete to moderate number of endometrial
glands in the light/dark and light groups, which were poorly developed in most animals,
except for one animal in the light group that presented well-developed endometrial
glands. Neovascularization in the lamina propria was observed in all fragments ([Fig. 2]).
Fig. 2 (A) Photomicrograph of the hemotrichorial placenta in a mouse of the light/dark group.
De: decidua; ZB: basal zone; ZL: labyrinth zone; PC: chorionic placenta; •: yolk sac;
* umbilical cord vessels. H&E, 2.5x. (B) Photomicrograph of the hemotrichorial placenta in a mouse of the light/dark group.
▪: Chorionic vessels in the chorionic plate. H&E,10x. (C) Photomicrograph of the hemotrichorial placenta in a mouse of the light/dark group.
Note the three types of cells in the basal layer: trophoblast giant cells (black arrow)
separating the basal zone (ZB) and decidua (De); glycogen cells (asterisk), and spongiotrophoblast
cells (red arrow). H&E., 10x. (D) Photomicrograph of the uterus in a mouse of the light/dark group. 1: Uterine lumen
where the dark line indicates the endometrium and the red line the myometrium; ▴:
placental tissue; 2: uterine tube. H&E., 2.5x. (E) Photomicrograph of the uterus in a mouse of the light/dark group. 1: endometrium
with a moderate number of glands. Note the eosinophilic content in the lumen of some
glands. 2: Myometrium. H&E., 2.5x. (F) Photomicrograph of the uterus in a mouse of the light/dark group. Arrow: simple
cylindrical epithelium; *: eosinophilic content in some glands. H&E, 20x.
Histological parameters of the chest and lungs of the fetuses were evaluated. Thorax
assessment revealed the presence of skin, muscle, cartilage, vertebral bodies, spine,
esophagus, trachea, thymus, heart, and lung in all groups. Pulmonary analysis showed
morphological features consistent with the transition from the canalicular to the
saccular phase in all animals. Only one animal from the light group exhibited tubuloacinar
structures in the absence of alveolar expansion and undifferentiated septal cells,
findings suggestive of the pseudoglandular phase ([Fig. 3]).
Fig. 3 (A) Photomicrograph of a cross-section of the fetal chest in the light/dark group. ▪:
Lung tissue; •: heart; *: esophagus; ▴: vertebral body; ♢: spine. H&E, 2.5x. (B) Photomicrograph of the fetal lung in the light/dark group. Black arrow: bronchioles;
red arrow: expansion of the alveolar sacs containing red blood cells and a moderate
amount of mesenchyme. H&E, 5x. (C) Photomicrograph of the fetal lung in the light/dark group. *: Bronchioles; •: esophagus.
(D) Photomicrograph of the fetal lung in the light/dark group. H&E, 20x. (E) Photomicrograph of the fetal lung in the light/dark group. Note the difference in
the amount of mesenchyme. H&E, 40x. (F) Photomicrograph of the fetal lung in the light/dark group. Arrows: type II pneumocytes.
Discussion
Considering the influence of environmental light, that is, light/dark cycle, on the
biological system, the findings for animals submitted to a light/dark period are in
concordance with the literature regarding litter size and average final female weight.
The litter of this study was composed by 113 pups, with an average of 13.6 pups per
female. An average of 8 to 10 pups per litter was reported in a study investigating
the control of reproduction in animal houses conducted in 2002.[8]
In the present study, the best average maternal variables were found for the light/dark
group. In this group, daytime and night-time periods were simulated, which influences
in an expected way the normal circadian rhythm of an individual who performs his/her
activities during the day and rests at night. The secretion of hormones and melatonin
follows the biological rhythm and does not affect the biological activities of the
organism.
The weight gain of females was higher in the light/dark group compared with the two
other groups, since the animals' normal routine was maintained in this group, with
melatonin secretion following the normal rhythm of the organism. The biological rhythm
of the animals was maintained close to normal. On the other hand, and in contrast
to the literature, a comparison of weight gain between the light and dark groups showed
a higher weight gain in animals deprived of light for 24 hours. Melatonin deprivation
or a reduction in its production has been shown to induce higher weight gain and can
possibly cause obesity.[9]
No histological placental alterations were observed in any of the groups, suggesting
that the exchange of nutrients after the placenta formation in pregnant mice was not
affected. On the other hand, histological analysis of the uteruses showed a reduction
in the number of endometrial glands in the light/dark and light groups. These glands
are necessary to provide adequate nutrition to the embryo, especially early on the
pregnancy, when the placental circulation is not fully established. Despite the alterations
in endometrial glands, the light/dark group gave birth to the largest litter of the
experiment.
The largest number of resorptions was observed in the dark group. Resorptions are
defined as the cessation of embryo development and are found after removal of the
uterus. They resemble the placenta but are smaller. Resorption can occur if the female
is exposed to a male pheromone that differs from the mating pheromone within 24 hours
after copulation.[8] The environment of our study was controlled to avoid such exposure. Thus, the resorptions
found were due to nutritional or structural deficits caused by the deregulation of
the circadian rhythm of the animals.
Melatonin receptors are found in the pineal gland, and also in other organs such as
the reproductive organs of humans and animals. Melatonin exerts action on the ovaries
and uterus and is also involved in placental implantation after mating.[10]
[11] Light exposure for short periods is unable to cause changes in maternal development.
Alterations have been reported when female mice are exposed to light for long periods
before mating.[11] In our study, females were exposed after mating, a fact that may explain the normal
development of pregnancy.
Alterations resulting from light exposure before mating, particularly morphological
changes, are caused by melatonin. These alterations mainly occur in the ovarian tissue,
leading to the development of polycystic ovaries in some females. In the uterus, the
changes are related to hypertrophy of the endometrial epithelium.[11]
Statistical analysis of fetal parameters showed a better development of almost all
parameters in the group exposed to light for 24 hours. This might be explained by
the longer period of maternal cortisol secretion. Since cortisol is regulated by the
circadian rhythm,[12] the peak production of this hormone depends on the presence of light, and lower
concentrations are thus observed when the animal is deprived of light. The passage
of maternal cortisol to the fetus throughout pregnancy is well-established.[13] Considering this maternal-fetal exchange, this pro-catabolic hormone can be related
to greater structural development of the offspring, since the fetuses would be more
exposed to its effects due to higher maternal secretion.
Microscopic analysis of the fetal specimens in all groups did not reveal major structural
alterations. Except for one animal of the light group that exhibited a delayed pulmonary
development, the parameters were similar in the remaining 133 fetuses. This finding
suggests that the exposure to different lighting conditions and the significant morphometric
alterations were not sufficient to cause changes at the cellular level in the animals
studied.
With these results, we can consider that shift factors must have attention to the
pregnant women health as an employment risk factor. Thus, expanding to bigger interests
in investigation in ambiance and health investigation, providing action plans for
prevention and health promotion.
Conclusion
The present results show that exposure to different lighting conditions during pregnancy
did not influence female reproductive parameters, while pups exposed to light throughout
pregnancy exhibited better morphometric measures. However, variations in luminosity
had no negative influence on the pregnancy of mice.
