Int. J. Morphol., 21(3):231-235, 2003.

ULTRASTRUCTURAL ANALYSIS OF PINEALECTOMY AND LACK OF LIGHT
INFLUENCE OVER COLLAGEN IN THE ENDOMETRIUM OF RATS

ANÁLISIS ULTRAESTRUCTURAL DE LA INFLUENCIA DE LA PINEALECTOMÍA
Y AUSENCIA DE LUZ SOBRE EL COLÁGENO EN EL ENDOMETRIO DE RATAS

^*Juliana Pinto de Medeiros; ^** Valéria Wanderley Teixeira; ^**Álvaro Aguiar Coelho Teixeira;
^***Liriane Baratella Evêncio & ^**Joaquim Evêncio Neto

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MEDEIROS, J. P.; WANDERLEY-TEIXEIRA, V.; TEIXEIRA, A. A. C.; BARATELLA-EVÊNCIO, L. & EVÊNCIO NETO, J. Ultrastructural analysis of pinealectomy and lack of light influence over collagen in the endometrium of rats. Int. J. Morphol., 21(3):231-235, 2003.

SUMMARY: Some studies have shown that pinealectomized female rats present a large concentration of collagen fibers in the endometrium. The lack of light results in morphologic alterations in the uterine lamina propria and in the muscular layer, reducing thus the uterine weight. Therefore, this work aimed to analyze ultra-structurally the influence of melatonin in the remodeling of the collagen in the endometrium, in the estrus phase, in pinealectomized adult rats or submitted to lack of light. The experimental groups: I- sham-pinealectomized maintained in a clear/dark cycle 12/12 hours, during three months; II- pinealectomized rats maintained in a clear/dark cycle 12/12 hours, for three months; III- normal rats maintained in clear/dark cycle 12/12 hours for three months; IV- normal rats maintained in lack of light, for three months. Fragments of a third of uterus average were gathered, fixed in Karnowsky solution and processed for Transmission Electron Microscope analysis. The results showed that the animals of the group II presented greater incidence of the estrus phase, in a percentile of 60%, and reduction of the proestrus phase. In the groups I, III and IV it had an equivalence of the estrus phase, however the animals of group IV showed a greater incidence of the diestrus phase with an average percentile of 40%. The ultra-structural analysis of the lamina propria of the endometrium in the animals of the groups I and III revealed several fibroblasts with nucleus presenting a little dense chromatin, and cytoplasm contents a great quantity of rugous endoplasmic reticulum. In the animals of the group II was observed a great quantity of fibroblasts, with several developed rugous endoplasmic reticulum, in spite of the great quantity of collagen fibers. In the animals of group IV themselves, few fibroblasts with small quantity of cytoplam that presents a scarce profile of rugous endoplasmic reticulum. Therefore, one can conclude that melatonin may perform a regulator factor in the collagen fibers synthesis by fibroblasts.

KEY WORDS: 1. Melatonin; 2. Endometrium; 3 Pinealectomy; 4. Collagen. 

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INTRODUCTION

Several authors report that pineal gland is the only endocrine gland directly influenced by external environment, through retinas pathways, where the environment signs are converted into neuroendocrine messages (Reiter, 1983; Binkley, 1993; Pevet, 1993). One knows that melatonin secretion regulation is related, in rodents, to the intensity and length of light exposure (Gauer et al., 1992). The biggest the luminous stimulus, smaller its synthesis, because exposure to light inhibits sympathetic fibers activities which innervate the pineal gland (Fritz & Speroff, 1982). According to Reiter (1991), melatonin is involved in neuroendocrine regulation of the reproduction in several species of mammals. The levels vary according to the different phases of the estral cycle of the rat (Johnson et al., 1982). Some studies have shown the influence of pinealectomy and lack of photoperiod over the feminine reproductive system. In relation to pinealectomy, Teixeira et al., (2002) reported a great concentration of collagen fibers in endometrium of pinealectomized rats. Schlatt et al., (1993) reported that when female hamsters are maintained in short photoperiods, between zero and 33 weeks, presents histological changes in ovary, although no significant changes have been observed in this gonad. Beyond this, these animals present a significant loss of uterine weight, between six and 27 weeks, in comparison to the ones of long photoperiod. Second these authors morphologic alterations in the uterus were observed in the lamina propria and in the muscular layer. Mendonça (2001), observed that the lamina propria of the endometrium of rats, maintained in lack photoperiod, presented itself poorly developed, showing more cellular than fibrous elements. Therefore, this work aimed to analyze ultra-structurally melanin influence in collagen of endometrium remodeling in estrus phase of pinealectomized adult rats or undergone to lack of light.

MATERIAL AND METHOD

Were utilized 24 albino rats (Rattus norvagicus albinus) by the Wistar lineage, with 90 days old, virgins, containning approximatelly 200g by weight and maintened in the biotery of the Morphology and Animal Phisiology Department of the Pernambuco Federal Rural University. The animals were kept in cages and maintained with ad libitum diet, at 22C with artificial lights by fluorescent lamps (Phillips make model day light, 40W), which established photoperiods of 12 hours clear and 12 hours dark, regarding the light period from 06:00am up to 06:00pm. After an adaptation period, vaginal varnish were gathered to determine regular estral cycle. The animals which presented regular estral cycles were submitted to surgery techniques of pinealectomy, and sham-pinealectomy, which were after divided, by chance in four groups:

Group I- sham pinealectomized rats maintained clear/dark of 12/12 hours cycle, for three months (control).
Group II- pinealectomized rats maintained in clear/dark of 12/12 hours cycle for three months.
Group III- normal rats maintained in clear/dark of 12/12 hours cycle for three months (control).
Group IV normal rats maintained in lack of light, for three months. The pinealectomy was performed in animals previously anesthetized with a pre anesthetic solution of atropine sulfate (0,1ml) diluted in 1ml of distilled water, being administrated 0,2ml of these in a intra-peritonial pathway, 10 minutes before the dissociate anesthesia, which was: 0,1ml of rompum (Xilazina ­ 20mg/ml) and 0,1ml) of Dopalen (Ketamine ­ 50mg/ml), intra-peritonial way. After that, the pinealectomy technique (Kuszak & Rodin, 1977) was proceeded. To the sham-pinealectomy, were performed all the proceedings mentioned above except the withdraw of pineal gland. The lack of light was obtained utilizing a 3m² room, totally sealed to not allow the entry of light, were the group IV animals were kept during the experiment. For ultra-structural analysis, the fragments of one third of the uterus was withdrawn, which was cut off and fixed in Karnowsky solution during four hours at environmental temperature, and after, maintained in freezer at 4C for 12 hours. After these proceedings, the fragments were taken to Pathology and Cellular Biology Lab at Ageu Magalhães Research Center at Pernambuco Federal University, to process the material and posterior analysis by aTransmission Electron Microscope, mark Jeol, model 100 CXII.

RESULTS

Colpocytological examination revealed that the animals of group II presented a greater incidence of estrum phase, with a percentile of 60%, comparing to other experimental groups. It was also observed in this group, that a decrease in proestrum phase. In the groups I, III and IV there was an equivalence in estrum phase, but animals of group IV showed a greater incidence in diestrum phase with an average percentile around 40%, related to the other experimental groups (Graphic I).

Graphic I. Frequency of the estrous cycle phase in the studied groups.

Ultra-structural analysis of the endometrial lamina propria of the animals sham-pinealectomized (group I) revealed several fibroblasts showing a little dense euchromatin and a great quantity of granular endoplasmatic reticulum in the cytoplasm. These results were similar to the ones observed in the animals of group III (Fig. 1). In pinealectomized animals (group II), was observed a large number of fibroblasts intercalated by several collagen fibers (Fig. 2) in the lamina propria of the endometrium. In the cytoplasm of these cells was verified the presence of a rich quantity of granular endoplasmic reticulum very developed (Fig. 3). The lamina propria of endometrium by the animals maintained in lack of light (group IV) revealed few fibroblasts, which presented scarce cytoplasm and a small quantity of granular endoplasmic reticulum (Fig. 4).

Fig. 1. Electronmicrography of a fibroblast (group I). Remark the nucleus (N) with a little dense chromatin. 30.000x.

Fig. 2. Electromicrography of lamina propria by endometrium of a rat of group II. Observe several fibroblasts (Arrows), intercalated by a great quantity of collagen fibers (C). 4.400x.

Fig. 3. Electronmicrography of lamina propria by the endometrium of the rat of group II. Observe great quantity of rugous emdoplasmic reticulum (RER) very developed. 30.000x.

Fig. 4. Electronmicrography of endometrial lamina propria of group IV. Observe collagen fibers (c) and a fibroblast (F) with a scarce cytoplasm. 7.000x.

DISCUSSION

It is known that in female rats, melatonin blood levels, vary according to the different phases of estral cycle (Reiter, 1993); and factors such as; age, season of the year, origin of the animals, as well feeding conditions and luminosity, that may provoke variations in the estral cycle of rats (Jaworski, 1950; Gronroos & Kauppila, 1959; Glickstein et al., 1972) The greatest incidence in estrus phase observed in the animals of group II, is accordingly to several researches which report that rats undergone to pinealectomy present low levels of melatonin in the bloodstream, consequently a greatest action of estrogen and androgen hormones (Chu et al., 1964; Chiorboly 1970; Teixeira et al., 2002), the animals of group IV presented a greater incidence in diestrus phase related to the other phases of estral cycle. This fact may be explained by the presence of high levels of melatonin in these animals, according to these results obtained by Cvijic et al., (1998) who observed similar behavior in rats maintained in constant dark during the period of six weeks.

Ultra-structural analysis revealed in the lamina propria of the endometrium, in estrus phase, of pinealectomized animals the presence of a great quantity of fibroblasts, which presented numerous rugous endoplasmic reticulum profiles, beyond of the great quantity of collagen fibers. In the animals maintained in lack of light few fibroblasts were observed, which presented a scarce cytoplasm and few quantity of granular endoplasmic reticulum. Simões et al., (1985) analyzed the ultra-structure of endometrium of rats during the estral cycle, reported that in the metaestrus and diestrus phases, the fibroblasts were more numerous comparing to other phases of cycle. these authors observed in these cells, the presence of several intra- cytoplasmatic vesicles containing collagen fibrils, showing thus, a collagen absorption activity in these phases of cycle. It is known that the levels of melatonin vary accordingly to different phases of the rats estral cycle, reaching the peaks during metaestrus and diestrus phases and decreasing in proestrus and estrus phases (Johnson et al., 1982). We still ought to mention that pineal gland withdraw in rats. It is followed by the increase of circulating estrogen, which stimulate the proliferation and activity of fibroblasts (Harkness et al., 1957; Bjersing & Borglin, 1964; Padykula & Campbell 1976; Benson & Orts, 1972; Simões et al., 1981 and Smaniotto, 1996).

Thus the presence of fibroblasts in a great synthesis activity is probably associate to the lack of melatonin and to the high levels of estrogens in the pinealectomized animals. In the animals maintained in lack of light, the presence of melatonin influenced in fibroblast activity making them to become inactive for the synthesis, so that, second Drobnik & Drabrowski (1996) the treatment with metalonin inhibits the increase of collagen. Therefore, we can conclude that melatonin can perform a regulatory activity in the synthesis of collagen fibers by fibroblasts. 

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MEDEIROS, J. P.; WANDERLEY-TEIXEIRA, V.; TEIXEIRA, A. A. C.; BARATELLA-EVÊNCIO, L. & EVÊNCIO NETO, J. Análisis ultraestructural de la influencia de la pinealectomía y ausencia de luz sobre el colágeno en el endometrio de ratas. Int. J. Morphol., 21(3):231-235, 2003.

RESUMEN: Algunos estudios han demostrado que ratas pinealectomizadas presentan gran concentración de fibras colágenas en el endometrio. La ausencia de luz resulta en alteraciones morfológicas en la lámina propia y capa muscular del útero, reduciendo el peso uterino. El objetivo de este trabajo fue analizar ultra-estructuramente la influencia de la melatonina en la remodelación del colágeno en el endometrio, en la fase de estro, en ratas adultas pinealectomizadas o sometidas a la ausencia de luz. Los grupos experimentales fueron: I ­ratas Sham-pinealectomizadas mantenidas en ciclo luz/oscuridad 12/12 horas, por tres meses; II ­ ratas pinealectomizadas mantenidas en ciclo luz/oscuridad 12/12 horas, por tres meses; III ­ratas normales mantenidas en ciclo luz/oscuridad 12/12 horas por tres meses; IV ­ ratas normales mantenidas en ausencia de luz, por tres meses. Se tomaron fragmentos del tercio medio del útero, fijados en solución de Karnowsky y procesados para análisis en microscopía electrónica de transmisión. Los resultados fueron: los animales del grupo II presentaron mayor incidencia de la fase de estro, con porcentaje de 60% y reducción de la fase de proestro al 4%. En los grupos I, III, y IV hubo una equivalencia de la fase de estro, sin embargo, los animales del grupo IV mostraron mayor incidencia de la fase de diestro con porcentaje alrededor de 40%. El análisis ultra-estructural de la lámina propia del endometrio de los animales de los grupos I y III reveló fibroblastos con núcleo con cromatina poco densa, y en el citoplasma gran cantidad de retículo endoplasmático rugoso. En los animales del grupo II, fue observada la presencia de gran cantidad de fibroblastos con numerosos retículos endoplasmáticos rugosos bastantes desarrollados, además de gran cantidad de fibras colágenas. En los animales del grupo IV, se observó pocos fibroblastos con escaso citoplasma y poca cantidad de retículo endoplasmático rugoso. Podemos concluir que la melatonina desempeña una actividad en la síntesis de fibras colágenas por los fibroblastos.

PALABRA CLAVE: 1. Melatonina; 2. Endometrio; 3. Pinealectomía; 4. Colágeno. 

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REFERENCES

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Teixeira, A. A.C.; Simões, M. J.; Evêncio Neto, J. & Wanderley-Teixeira, V. Morphologic aspects of the endometrium, in the estrus phase, of pinealectomized rats. Rev. Chil. Anat., 20(2):145-9, 2002.

Correspondence to:
Profa. Dra. Valéria Wanderley Teixeira.
Universidade Federal Rural de Pernambuco.
Departamento de Morfologia e Fisiologia Animal.
Área de Histologia
Rua Dom Manoel de Medeiros s/n ­ Dois Irmãos
CEP: 52.171-900
Recife-PE. BRAZIL

e-mail: vwt@bol.com.br

Received : 30-06-2003
Accepted : 30-07-2003 

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^* Veterinary Medicine, Universidade Federal Rural de Pernambuco, Recife-PE, Brazil.
^** Department of Morphology and Animal Physiology, Universidade Federal Rural de Pernambuco, Recife - PE, Brazil.
^*** Department of Histology and Embriology, Universidade Federal de Pernambuco - PE, Brazil.