Acta anal. 02: 1-7 (1975)
Department of Morphology, Faculty of Medicine of Riheirao Prcto. University of Sao Paulo, Sao Paulo, Brazil
Mitotic activity ami nuclear/cytoplasmatic ratio of the lachry mal and Harderian glands of the rat during postnatal life G ekaldo F. Vianna , A ndre R .C ruz and R einaldo A zoubel Key Words. Mitotic activity • Nuclear/cytoplasmatic ratio • Lachrymal and Harderian glands
Introduction In a previous study, Vianna el til. [1974) observed that the Harderian and lach rymal glands of the rat presented a two-stage differential growth, the first from birth until the 15th day of postnatal life, and the second from the 15th day until the final period of life studied. These two stages of growth were separated by a critical period, during which an abrupt change occurred in the value of the allomclric coefficient. This critical period, observed in the glands around the 15th day of postnatal life, had also been noticed in other organs by Cruz |1962|. I ucif [1962], Cruz and P ortioli [1963], AzOUBEL [1964], and in many other fields and aspects by Sesso 11962], Sesso et til. |1962. 1965], and Oliverio [1965], In the present work, the authors studied the mitotic activity and the nuclear/cytoplasrnic (N/C) ratio relationships in the lachrymal and Harderian glands of the rat during postnatal life, in an attempt to evaluate the structural alterations which might justify the modifications in growth rhythm observed.
For the evaluation of mitotic activity, 66 rats (Rattus norvergicus alhinus) were divided into two groups: one included animals up to the 40th day of postnatal life, the other animals having passed this period. 76 rats were used to determine the N/C ratio of the lachrymal and Harderian glands of the right side. Weighing and dissection. The rats were sacrificed by cranial trauma and decapi tated. The heads were fixed in an Alfac solution (80 % alcohol, 85 ml: formalin, 10 ml: acetic acid, 5 ml) for 24 h. The glands were dissected following the instructions of Venable and Grafflin [1940] with the help of a binocular magnifier (Zeiss, x40) and returned for a further 24 li of fixation. Histological technique. After fixation the glands were embedded in paraffin and slices of 6 /mi were obtained. The coloration techniques utilized were those of F eulgen and Roszenberg [1924J with 18 min of hydrolysis [Lison, I960], for mitosis counts and Masson’s trichrome stain to evaluate N/C relations. To evaluate
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Material and methods
2
V ia nn a el at. Mitotic activity and nuclear/cytoplasmatic ratio
mitotic activity of the lachrymal and Harderian glands, 1,000 cells were counted; those presenting cellular division were selected. A screw micrometer reticulate eyepiece was utilized with an (X 100) immersion objective. The N/C ratio was evaluated by Chai.kley’s [19431 technique, modified by E ranko [1955]. The original arrows were replaced by a reticulum, constituting eleven perpendicular lines and structures coincident with 117 points. 1,500 points were counted and the elements of 13 microscopic fields registered. The cytological elements considered were: cytoplasm, nucleus, lumen of acini and ducts, all other tissular components and the 'point behind focus’ were considered 'remaining structures’.
Results
Fig. I. Mitotic activity of lachrymal (--------) and Harderian (---------) glands. The points correspond to the mean of the values of each age group.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Mitotic activity o f the lachrymal ami Harderian "lands during postnatal life After birth, the H arderian and lachrym al glands of the rat ex hibited a high m itotic activity. The analysis of the graphic projection (fig. 1) of the num ber of mitoses by 1,000 cells, in each age group, dem onstrated th a t cellular m ultiplication decreased gradually until the 20th day of postnatal life. In the adult animals, although figures of mitosis were onlv rarely observed, this cellular capacity did not disappear com pletely. M itotic activity was slightly higher for the H arderian than for the lachrym al gland during the 1st day of postnatal life. However, from
of tlie lachrymal and Harderian glands of the ral during postnatal life
3
the 8tli day of postnatal life on, both glands behaved in a similar way. Between the 10th and 20th day a rapid dim inution of the cellular division process was observed. During this period, a reduc tion to less than half of the mean rates observed in the first fortnight was observed, the rate for the lachrym al gland falling front 12.20 to 3.85, and for the H arderian gland from 11.00 to 3.28.
Fig. 2. Graphic representation of the mean (A, A) of the N/C relation of individual ( • . O) values in each age group.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Nuclear cytoplasmatic ratios The N/C ratio of lachrym al and Harderian glands, was unity during the 1st day of postnatal life, the mean values for the newborn rats being 1.54 and 1.31, respectively (fig.2). From the 5th day of postnatal life until the 15th day, the values of this relation grew progressively slower. From then on, the increase was more rapid and after the 40th day rem ained constant until the final period of life studied. Figure2 indicates t hat t he two glands exhibited different behaviour from the 25th day onwards, the increase in the value of the N/C relation being more rapid for the H arderian gland. At the 40th «lay, both curves, th a t for the H arderian gland now located at a higher level, presented a certain degree of stability or constancy, the variations around the average being more evident for
4
V ianna et al. Mitotic activity and nuclear cytoplasmatic ratio
Discussion The period of grow th of the lachrym al and Harderian glands was coincident with the tim e at which the evelids opened |V ianna el til.. 19741. In some species the eyelid disjunction occurs at a late stage of fetal life and in others only after birth. The time of this opening is related to general developm ent, and in particular with th a t of the retinae [A ddison and How, 19211. From the 18lh day of fetal life, the rat's eyelids blend and they remain united until the 15th day of postnatal life. During the first stage of observation, intense mitotic activity of the lachrym al and H arderian glands was observed; on the first day of life, the m itotic activity of the lachrvm al gland was slightly sm aller than th a t of the H arderian gland. At the la th day, m itotic activitv of both glands fell to its lowest rate, corroborating the results obtained by T eir 11944, 1952] for the lachrym al gland. The period at which the decrease in cellular division occurred coinci ded with the “crisis" period of the rat's developm ent. This period is characterized in the curve of ailom clric growth, at a critical point already analyzed by V i anna el ul . | 1974]. This agreem ent was also observed by S hade |1959| and A zovbel |19(>4| for the m itotic activity in the cerebral cortex and retina, respectively. In the present study m itotic activity was defined b\ the num ber of mitotic figures present in 100 150 microscopic fields (40 X objec tive and 10 X ocular). Since the lachrym al gland presents verv precocious nuclear classes, the num ber of cells in each microscopic field decreased with the developm ent of the animal. While a com plete loss of the capacity of cellular m ultiplication was not observed, a great reduction in this capacity was noted in both glands. Exceptional figures of mitosis in adult anim als were observed. W ai.kek 11958], studied the process of cellular division in the lachrym al gland of the rat after the 21st day of postnatal life. As dem onstrated in the present work, the m itotic activity in this phase is at very low levels, in contrast to the previous, exuberant phase. Our observations agree with the results of T eir 11944, 1952]. Vianna el til. 11974] observed th at the two-step developm ent of the
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
this gland. In figure 2, we can observe th a t only one of the values of the N/C ratio of the H arderian gland coincided with the area representing the maximum and m inim um ol' the individual values obtained for the lachrym al gland.
lachrym al anti H arderian glands of the ral is not different, indicating that both glands grow quickly until the 15th day of life. Two mechanisms seem to be im plicated: the more rapid initial growth by successive events would lead to a definitive functional differentiation of the organs. This mechanism is represented by the phase of intense cellular m ultiplication, during which growth occurs only by the increase in the num ber of cells, and an N/C ratio of around unity. The second mechanism, represented by cellular differentiation, is characterized by cytoplasm atic enlargement and a fall in cellular division. During this period the growth persists not only through an increase in the num ber of cells but also by an increase in the size of the cells themselves. This hypothesis, already discussed in relation to mitosis, is now supported by the behaviour of the N C relation. During the 1st week this value increased slowly and progressively, so th at 1 week before eyelid-opening an abrupt increase was observed, remaining until a plateau was reached at 40 days. This phase of rapid increase of the N/C relation would characterize cell differentiation. The Harderian gland presented a different behaviour in relation to the lachrym al gland, always showing higher values. Sesso et nl. [1962], studying the postnatal developm ent of the pancreas, analyzed the allom etric relation of RN AP DNAP of pancreas homogenates. Three phases were defined, the second being characterized by a more elevated value of k (k = 1.078) in the period of 15 28 day's of life, indicating an accum ulation of RNA bv the cells. In the first step, a reduction in the qu antity of RNA (k = 0.842) occurred; in the third phase, an accum ulation, not so evident as in the growing phase (k 1.171) was observed. Similar observations were made in relation to the grow th of the pancreas, w ith three stages of differentiation [S esso et til.. 1965]. The rapid growth phase of this organ continued until the 30th day, being also characterized bv an increase in the total num ber of cells, estim ated by the rate of DNA increase. The different location of critical points, between 15 and 26 days in the case of the pancreas, and on the 15th day for the glands in the present study, could be explained by the different functional requirem ents of these organs. This conclusion could indicate, sim ilarly to what has been previously observed, the existence of the same type of relations between cellular division, modification of the cytoplasm atic nuclear ratio and the developm ent of the organ.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
of the lachrymal and Harderian glands of the rat during postnatal life
6
V ianna el ill. Mitotic activity and nuelear/cytoplasinatie ratio
Sum inary The autliors performed a study of the mitotic activity and the nuclear cyto plasmic (N/C) ratio during postnatal life of the lachrymal and Ilardcrian glands of the rat. Based on the results, they concluded: (1) during the first days of postnatal life the development of lachrymal and Ilardcrian glands was characterized by an intense mitotic activity and a low N C ratio: (2) the period prior to eyelid disjunct ion was characterized by a diminished mitotic activity and a progressive and slow increase of the N/C ratio: (3) after eyelid disjunction, mitotic activity was reduced and an abrupt increase of the N/C ratio occurred, more evident in the Ilardcrian gland; (4) during the final period of postnatal life studied mitotic activity was ab sent and the N/C ratio presented a higher, more constant level, which was always higher for the Ilardcrian gland.
References completion of disjunction. Anter. .1. Anal. 20: 1 il (1921). Azot HK1-. 1C: Crescimenlo alométrico do bulbo ocular durante o desenvolvimento ontogenético da galinha e do rato: thesis Ribcirao Proto (1964). Ciialkley. II. W .: Method for the quantitative morphologic analysis of tissues. J. nal. Cancer Inst. 4: 47-54 (1943). Criz . A. R .: Kstudo do crescimenlo pós-natal do testículo e da supra renal do rato. A rcla
Department of Morphology, Faculty of Medicine of Riheirao Prcto. University of Sao Paulo, Sao Paulo, Brazil
Mitotic activity ami nuclear/cytoplasmatic ratio of the lachry mal and Harderian glands of the rat during postnatal life G ekaldo F. Vianna , A ndre R .C ruz and R einaldo A zoubel Key Words. Mitotic activity • Nuclear/cytoplasmatic ratio • Lachrymal and Harderian glands
Introduction In a previous study, Vianna el til. [1974) observed that the Harderian and lach rymal glands of the rat presented a two-stage differential growth, the first from birth until the 15th day of postnatal life, and the second from the 15th day until the final period of life studied. These two stages of growth were separated by a critical period, during which an abrupt change occurred in the value of the allomclric coefficient. This critical period, observed in the glands around the 15th day of postnatal life, had also been noticed in other organs by Cruz |1962|. I ucif [1962], Cruz and P ortioli [1963], AzOUBEL [1964], and in many other fields and aspects by Sesso 11962], Sesso et til. |1962. 1965], and Oliverio [1965], In the present work, the authors studied the mitotic activity and the nuclear/cytoplasrnic (N/C) ratio relationships in the lachrymal and Harderian glands of the rat during postnatal life, in an attempt to evaluate the structural alterations which might justify the modifications in growth rhythm observed.
For the evaluation of mitotic activity, 66 rats (Rattus norvergicus alhinus) were divided into two groups: one included animals up to the 40th day of postnatal life, the other animals having passed this period. 76 rats were used to determine the N/C ratio of the lachrymal and Harderian glands of the right side. Weighing and dissection. The rats were sacrificed by cranial trauma and decapi tated. The heads were fixed in an Alfac solution (80 % alcohol, 85 ml: formalin, 10 ml: acetic acid, 5 ml) for 24 h. The glands were dissected following the instructions of Venable and Grafflin [1940] with the help of a binocular magnifier (Zeiss, x40) and returned for a further 24 li of fixation. Histological technique. After fixation the glands were embedded in paraffin and slices of 6 /mi were obtained. The coloration techniques utilized were those of F eulgen and Roszenberg [1924J with 18 min of hydrolysis [Lison, I960], for mitosis counts and Masson’s trichrome stain to evaluate N/C relations. To evaluate
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Material and methods
2
V ia nn a el at. Mitotic activity and nuclear/cytoplasmatic ratio
mitotic activity of the lachrymal and Harderian glands, 1,000 cells were counted; those presenting cellular division were selected. A screw micrometer reticulate eyepiece was utilized with an (X 100) immersion objective. The N/C ratio was evaluated by Chai.kley’s [19431 technique, modified by E ranko [1955]. The original arrows were replaced by a reticulum, constituting eleven perpendicular lines and structures coincident with 117 points. 1,500 points were counted and the elements of 13 microscopic fields registered. The cytological elements considered were: cytoplasm, nucleus, lumen of acini and ducts, all other tissular components and the 'point behind focus’ were considered 'remaining structures’.
Results
Fig. I. Mitotic activity of lachrymal (--------) and Harderian (---------) glands. The points correspond to the mean of the values of each age group.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Mitotic activity o f the lachrymal ami Harderian "lands during postnatal life After birth, the H arderian and lachrym al glands of the rat ex hibited a high m itotic activity. The analysis of the graphic projection (fig. 1) of the num ber of mitoses by 1,000 cells, in each age group, dem onstrated th a t cellular m ultiplication decreased gradually until the 20th day of postnatal life. In the adult animals, although figures of mitosis were onlv rarely observed, this cellular capacity did not disappear com pletely. M itotic activity was slightly higher for the H arderian than for the lachrym al gland during the 1st day of postnatal life. However, from
of tlie lachrymal and Harderian glands of the ral during postnatal life
3
the 8tli day of postnatal life on, both glands behaved in a similar way. Between the 10th and 20th day a rapid dim inution of the cellular division process was observed. During this period, a reduc tion to less than half of the mean rates observed in the first fortnight was observed, the rate for the lachrym al gland falling front 12.20 to 3.85, and for the H arderian gland from 11.00 to 3.28.
Fig. 2. Graphic representation of the mean (A, A) of the N/C relation of individual ( • . O) values in each age group.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
Nuclear cytoplasmatic ratios The N/C ratio of lachrym al and Harderian glands, was unity during the 1st day of postnatal life, the mean values for the newborn rats being 1.54 and 1.31, respectively (fig.2). From the 5th day of postnatal life until the 15th day, the values of this relation grew progressively slower. From then on, the increase was more rapid and after the 40th day rem ained constant until the final period of life studied. Figure2 indicates t hat t he two glands exhibited different behaviour from the 25th day onwards, the increase in the value of the N/C relation being more rapid for the H arderian gland. At the 40th «lay, both curves, th a t for the H arderian gland now located at a higher level, presented a certain degree of stability or constancy, the variations around the average being more evident for
4
V ianna et al. Mitotic activity and nuclear cytoplasmatic ratio
Discussion The period of grow th of the lachrym al and Harderian glands was coincident with the tim e at which the evelids opened |V ianna el til.. 19741. In some species the eyelid disjunction occurs at a late stage of fetal life and in others only after birth. The time of this opening is related to general developm ent, and in particular with th a t of the retinae [A ddison and How, 19211. From the 18lh day of fetal life, the rat's eyelids blend and they remain united until the 15th day of postnatal life. During the first stage of observation, intense mitotic activity of the lachrym al and H arderian glands was observed; on the first day of life, the m itotic activity of the lachrvm al gland was slightly sm aller than th a t of the H arderian gland. At the la th day, m itotic activitv of both glands fell to its lowest rate, corroborating the results obtained by T eir 11944, 1952] for the lachrym al gland. The period at which the decrease in cellular division occurred coinci ded with the “crisis" period of the rat's developm ent. This period is characterized in the curve of ailom clric growth, at a critical point already analyzed by V i anna el ul . | 1974]. This agreem ent was also observed by S hade |1959| and A zovbel |19(>4| for the m itotic activity in the cerebral cortex and retina, respectively. In the present study m itotic activity was defined b\ the num ber of mitotic figures present in 100 150 microscopic fields (40 X objec tive and 10 X ocular). Since the lachrym al gland presents verv precocious nuclear classes, the num ber of cells in each microscopic field decreased with the developm ent of the animal. While a com plete loss of the capacity of cellular m ultiplication was not observed, a great reduction in this capacity was noted in both glands. Exceptional figures of mitosis in adult anim als were observed. W ai.kek 11958], studied the process of cellular division in the lachrym al gland of the rat after the 21st day of postnatal life. As dem onstrated in the present work, the m itotic activity in this phase is at very low levels, in contrast to the previous, exuberant phase. Our observations agree with the results of T eir 11944, 1952]. Vianna el til. 11974] observed th at the two-step developm ent of the
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
this gland. In figure 2, we can observe th a t only one of the values of the N/C ratio of the H arderian gland coincided with the area representing the maximum and m inim um ol' the individual values obtained for the lachrym al gland.
lachrym al anti H arderian glands of the ral is not different, indicating that both glands grow quickly until the 15th day of life. Two mechanisms seem to be im plicated: the more rapid initial growth by successive events would lead to a definitive functional differentiation of the organs. This mechanism is represented by the phase of intense cellular m ultiplication, during which growth occurs only by the increase in the num ber of cells, and an N/C ratio of around unity. The second mechanism, represented by cellular differentiation, is characterized by cytoplasm atic enlargement and a fall in cellular division. During this period the growth persists not only through an increase in the num ber of cells but also by an increase in the size of the cells themselves. This hypothesis, already discussed in relation to mitosis, is now supported by the behaviour of the N C relation. During the 1st week this value increased slowly and progressively, so th at 1 week before eyelid-opening an abrupt increase was observed, remaining until a plateau was reached at 40 days. This phase of rapid increase of the N/C relation would characterize cell differentiation. The Harderian gland presented a different behaviour in relation to the lachrym al gland, always showing higher values. Sesso et nl. [1962], studying the postnatal developm ent of the pancreas, analyzed the allom etric relation of RN AP DNAP of pancreas homogenates. Three phases were defined, the second being characterized by a more elevated value of k (k = 1.078) in the period of 15 28 day's of life, indicating an accum ulation of RNA bv the cells. In the first step, a reduction in the qu antity of RNA (k = 0.842) occurred; in the third phase, an accum ulation, not so evident as in the growing phase (k 1.171) was observed. Similar observations were made in relation to the grow th of the pancreas, w ith three stages of differentiation [S esso et til.. 1965]. The rapid growth phase of this organ continued until the 30th day, being also characterized bv an increase in the total num ber of cells, estim ated by the rate of DNA increase. The different location of critical points, between 15 and 26 days in the case of the pancreas, and on the 15th day for the glands in the present study, could be explained by the different functional requirem ents of these organs. This conclusion could indicate, sim ilarly to what has been previously observed, the existence of the same type of relations between cellular division, modification of the cytoplasm atic nuclear ratio and the developm ent of the organ.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/16/2019 6:28:52 AM
of the lachrymal and Harderian glands of the rat during postnatal life
6
V ianna el ill. Mitotic activity and nuelear/cytoplasinatie ratio
Sum inary The autliors performed a study of the mitotic activity and the nuclear cyto plasmic (N/C) ratio during postnatal life of the lachrymal and Ilardcrian glands of the rat. Based on the results, they concluded: (1) during the first days of postnatal life the development of lachrymal and Ilardcrian glands was characterized by an intense mitotic activity and a low N C ratio: (2) the period prior to eyelid disjunct ion was characterized by a diminished mitotic activity and a progressive and slow increase of the N/C ratio: (3) after eyelid disjunction, mitotic activity was reduced and an abrupt increase of the N/C ratio occurred, more evident in the Ilardcrian gland; (4) during the final period of postnatal life studied mitotic activity was ab sent and the N/C ratio presented a higher, more constant level, which was always higher for the Ilardcrian gland.
References completion of disjunction. Anter. .1. Anal. 20: 1 il (1921). Azot HK1-. 1C: Crescimenlo alométrico do bulbo ocular durante o desenvolvimento ontogenético da galinha e do rato: thesis Ribcirao Proto (1964). Ciialkley. II. W .: Method for the quantitative morphologic analysis of tissues. J. nal. Cancer Inst. 4: 47-54 (1943). Criz . A. R .: Kstudo do crescimenlo pós-natal do testículo e da supra renal do rato. A rcla
