[EN]Lactation is a physiological process associated
with hyperactivity of hypophyseal prolactin-producing
cells. It is known that the percentage of these cells is increased
during lactation, although there are discrepancies
in the reports regarding the mechanisms responsible for
increasing the number of prolactin cells. In order to analyse
whether this increase is a result of previous proliferation,
variations in the proliferation rate of prolactinpositive
cells were determined from late pregnancy to lactation
in adult female rats by means of observation of the
immunohistochemical expression of PCNA as a marker
of cellular proliferation. During late pregnancy, a very
significant increase in the percentage of proliferating prolactin
cells was observed in comparison to non-pregnant
females in the proestrus phase (p < 0.01). Although the
percentage of prolactin-positive cells after one week of
lactation was higher than in non-lactating or in pregnant
females (p < 0.01), the proliferation rate was lower than
in the other groups studied. In sum, our results suggest
that late pregnancy constitutes a preliminary proliferative
phase preparatory to the ensuing lactation phase and that
endocrine changes in late pregnancy involve the cellular
proliferation of hypophyseal prolactin cells in order to
prepare the gland for later demands and to prevent proliferative
changes from occurring during lactation

Blanco, Enrique

Bodego, Pilar

Burks, Deborah J.

Carretero González, José

Hernández, Elena

Juanes Méndez, Juan Antonio

Riesco-López, José M.

Rubio, Manuel

Torres, José L.

Vázquez, Ricardo

Annals of Anatomy - Anatomischer Anzeiger

English

Gestion del Repositorio Documental de la Universidad de Salamanca

ANNALS OF ANATOMY Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats Jos6 Carretero, Manuel Rubio, Enrique Blanco, Deborah J. Burks, Jos6 L. Torres, Eiena Hernfindez, Pilar Bodego, Jos6 M. Riesco, Juan A. Juanes, and Ricardo Vfizquez Laboratorio de Neuroendocrinologfa, Instituto de Neurociencias de Castilla y Le6n, Departamento de Anatomfa e Histologfa Humanas, Facultad de Medicina, Universidad de Salamanca, Avda. Alfonso X E1 Sabio s/n, 37007 Salamanca, Spain Summary. Lactation is a physiological process associated with hyperactivity of hypophyseal prolactin-producing cells. It is known that the percentage of these cells is in- creased during lactation, although there are discrepancies in the reports regarding the mechanisms responsible for increasing the number of prolactin cells. In order to ana- lyse whether this increase is a result of previous prolifera- tion, variations in the proliferation rate of prolactin- positive cells were determined from late pregnancy to lac- tation in adult female rats by means of observation of the immunohistochemical expression of PCNA as a marker of cellular proliferation. During late pregnancy, a very significant increase in the percentage of proliferating pro- lactin cells was observed in comparison to non-pregnant females in the proestrus phase (p < 0.01). Although the percentage of prolactin-positive cells after one week of lactation was higher than in non-lactating or in pregnant females (p < 0.01), the proliferation rate was lower than in the other groups studied. In sum, our results suggest that late pregnancy constitutes a preliminary proliferative phase preparatory to the ensuing lactation phase and that endocrine changes in late pregnancy involve the cellular proliferation of hypophyseal prolactin cells in order to prepare the gland for later demands and to prevent pro- liferative changes from occurring during lactation. Key words: Prolactin cells - Cellular proliferation - Late pregnancy - Lactation Correspondence to: J. Carretero E-mail: jcar@usal.es Introduction Prolactin is a hypophyseal hormone involved in the regu- lation of lactation and its release is increased during this physiological process (Meites and Turner 1948; Meites et al. 1972; Cowie et al. 1980). Within the first days and weeks postpartum, basal prolactin levels remain elevated and suckling episodes trigger a rapid release of hypophy- seal prolactin (Tyson and Friesen 1973; Noel et al. 1974). This increase in prolactin secretion is accompanied by a suckling-induced increase in hypophyseal prolactin mRNA levels (Lee et al. 1989). Basal prolactin levels gradually increase throughout the course of pregnancy (Tyson and Friesen 1973; Rigg et al. 1977; Ben-David et al. 1973). This has been attributed to the stimulatory effect of estrogens on hypophyseal lacto- trophs. When hypophyseal prolactin synthesis and secre- tion are stimulated the lactotroph population increases. It has been suggested that this increase could be due to the differentiation of somatotroph to lactotroph cells (Boock- for et al. 1987) or to the proliferation of lactotroph cells (Lloyd et al. 1975; Kalbermann et al. 1979; Perez et al. 1986). A gradual increase in the number of hypophyseal lactotrophs during pregnancy has been reported (Golub- off and Ezrin 1969; Scheithauer et al. 1990). However, the proliferative mechanisms involved in this increase re- main obscure. The aim of the present study was to determine whether cellular proliferation is involved in the increase in the percentage of prolactin cells during late pregnancy and lactation and to determine whether proliferation occurs during both late pregnancy and lactation. Ann Anat (2003) 185:97-101 © Urban & Fischer Verlag http:llwww.urbanfischer.deljournalslannanat 0940-9602/03/185/2-97 $15.00/0 Materials and methods  Animals. Fifteen female adult Sprage-Dawley rats (200 g b/w) were used and were kept under standard conditions (22 + 2°C, RH: 50 + 5%, 8.00 to 20.00 light hours), with water and food (Panlab ® maintenance diet) ad libitum. Animals were handled (cleaning, handling, hygiene) according to the guidelines of the European Communities Council Directive (86/609/EEC) and cur- rent Spanish legislation for the use and care of laboratory ani- mals (BOE 67/8509-12,1998). The rats were divided into 3 experimental groups (5 animals per group): (1) untreated rats in the proestrous phase, as deter- mined by vaginal smears, were considered as normal and em- ployed as controls; (2) rats sacrified after 1 week of suckling in their respective litters; (3) animals killed on day 19 of pregnancy. Sacrifice of animals and sample processing. The animals were sacrificed by decapitation following anesthesia by Forene inhala- tion. The hypophyses were carefully dissected out and immedi- ately fixed in a solution of 15% saturated pricric acid in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 24 h. Then, they were dehydrated in ethanol, cleared with xylene, and embedded in paraffin in order to obtain coronal serial sections of 5 gm thickness. These were placed on slides treated with gela- tin-chrome alum and were then used for the inmunohistochem- ical study. Immunohistochemistry. To study PCNA-positive cells and to determine the PCNA-Prolactin labelling index, a double label- ling immunohistochemical method for PCNA and prolactin was developed. Endogenous peroxidase was blocked with H202 in methanol and non-specific reactions of the secondary antibody by incubation in normal goat serum (Dako, diluted 1 : 30). Sec- tions were incubated overnight at 4 °C with mouse anti-PCNA monoclonal antibody (PC 10, Dako, diluted 1:3 000 in TBS). Biotinylated goat anti-mouse IgG (Dako, diluted 1:100) and Avidin-Biotinylated horseradish peroxidase complex (ABC kit, Dako, diluted 1:100) were successively applied at room tem- perature for 40 min and 30 min, respectively. The reaction was developed in freshly prepared 3,3'-DAB (0.025% in TRIS buffer containing 0.03% of H2Oz). Following PCNA immunola- belling, the peroxidase-antiperoxidase (PAP) reaction was per- formed for the detection of prolactin, using as primary serum anti-prolactin rabbit serum at a dilution of 1:800, swine anti- rabbit serum (Dako, diluted 1:100), and rabbit-PAP complex (Dako, diluted 1:100). Preabsorption tests with Prolactin and tests substituting the specific serum by normal rabbit serum abolished the reaction. Using ELISA, the specificity of swine anti-rabbit IgG was lower than 1% for rat and mouse IgG and 100% for rabbit IgG. For the washes and dilutions of the sera, TRIS buffer (0.05 M, pH 7.4) containing 0.8% NaC1 was used. The reaction was developed in freshly prepared 4-chloro-1- naphthol (1.7 x 10 -3 M in 3% absolute ethanol and TRIS-buffer containing 0.3% H202). Prolactin- and PCNA-prolactin-immunoreactive cell quantifica- tion. Four thousand cells per animal were evaluated using a Leitz Dialux EB-20 microscope at a final magnification of 500 x. The cells were randomly selected from different areas of the gland. The following parameters were determined: 1) PCNA-positive cells; 2) Prolactin-positive cells; 3) PCNA- and prolactin-positive cells (always calculated as percentages of the total number of cells analyzed). The percentage of PCNA- and prolactin-immu- noreactive cells was also calculated from the total of prolactin- immunoreactive cells. Statistical analysis. For each parameter evaluated, the values obtained were processed statistically and the differences observed were compared using analysis of variance, accepting p values of < 0.05 as significant for the Scheff6 F test. Results are expressed as arithmetic means _+ standard error of the mean. Results Hypophyseal proliferation. Hypophysea l  prol i fera t ion,  de- t e rmined  as the  percen tage  of cells with P C N A - i m m u n o -  react ive nuclei, was low in females in proes t rous  (0.74_+ 0.05). In late pregnancy, a significant increase in cells undergoing  pro l i fe ra t ion  was observed:  9.41 +_ 0.79% of g landular  cells were  P C N A  posi t ive (p < 0.01 in com- par i son  to nonpregnan t  females) .  In  suckling females,  the percen tage  of prol i fera t ing  cells was s imilar  to normal  animals  (0.55 + 0.09 vs. 0.74 + 0.05) and significantly lower (p < 0.01) than that  observed  in p regnant  females. Protactin-positive ceil proliferation. Pro lac t iu -PCNA-  posi t ive cells in females in the proes t rous  phase  were 0.44 + 0.01%. This means  that  1.46% of the  prolac t in  cells were  prol i fera t ing cells. In  these  animals  30.22 + 0.28% of the  g landular  cells were prolact in-posi t ive.  Pregnancy was seen to be an impor tan t  prol i fera t ion-  inducing agent  in prolact in-posi t ive  cells. In  the  p regnant  animals  4.00 _+ 0.51% of all cells were  p ro lac t in -PCNA-  positive, and hence 14.06% of the prolac t in  cells were prol i fera t ing cells during late pregnancy. Al though  the 12 10 .>_ 8 0 < z (9 6 ca 4 e-- I l l  ca I D Proes t rous  [] Ges ta t ion  [] Lactat ion I Fig. 1. Plot showing the mean values of the percentage of the percentage of PCNA-positive cells in the different groups stud- ied. 9 8  percentage of prolactin-positive cells was lower than in proestrous females, the observed statistical differences were not significant (28.44 + 0.90 vs. 30.22 + 0.28). The percentage of proliferating prolactin-positive cells in suckling females was very low, since 0.22 + 0.08% were both prolactin- and PCNA-positive cells (p < 0.05 in rela- tion to the proestrous animals and p < 0.01 in relation to the pregnant females). Thus, during suckling only 0.49% of prolactin-positive cells were undergoing proliferation. 50 = 4 5  ._~ 40 35 ~, a0 t9 25 £1. 20 ~ 1 5  e.- o • 10 i -  o. 5 0 [[] Proestrous [] Gestat ion [] Lactation ] Fig. 2. Plot showing the mean values of the percentage of prolac- tin-positive cells in the different groups studied. 4,5 • ~ 4 :~ 3,5 t/l O ,o. '~ 3 z t.9 0. 2,5 t~ 2 t9 t~ a. 1,5 O ¢ o ~. 0,5 [[] Proestrous [] Gesta t ion  [] Lactat ion ] Fig. 3. Plot showing the mean values of the percentage of PCNA- and prolactin-positive cells in the different groups stud- ied. Comparatively, the percentage of prolactin-immuno- reactive cells was higher in suckling females than in the animals in proestrus (44.67+ 1.38 vs. 30.22+0.28, p < 0.01) or late pregnancy (44.67 + 1.38 vs. 28.44 + 0.90, p < 0.01). /+ Fig. 4. Micrographs showing the immunostain for PCNA (brown) and/or prolactin (dark blue) in proestrus female rats (4 a: x 100, 4 b: x 300), pregnant female rats (4 c: x 100, 4 d: x 300) and lactating female rats (4 e: x 100, 4 f: x 300). 99 Discussion It is conventionally accepted that the hypophyseal cellular proliferation rate is very low in adult animals (Pomerat 1941; Hunt  1943; St~dtler et al. 1970; Stepi6n et al. 1978). Oishi et al. (1993) reported important variations related to the different phases of estrual cycle and lactotroph cells were found to show the highest proliferation rate of all types of hypophyseal cells. In agreement with the find- ings reported by Oishi and co-workers, our results de- monstrate a low cellular proliferation in female rats during the proestrous phase. The cellular proliferation of lactotroph cells has mainly been analysed following treatment with estradiol and do- pamine antagonists or in tumor cells (Lloyd et al. 1975; Kalbermann et al. 1979; P6rez et al. 1986; Stefaneanu et al. 1992; Oishi et al. 1993; Carretero et al. 1995). Except during postnatal development (Carbajo-Pdrez y Watanabe 1990), the proliferation of lactotroph cells de- creases progressively in relation to age (Takahashi et al. 1984). However, the proliferation of lactotroph cells is very important for the maintenance of total hypophyseal proliferation because 60.6% of proliferating cells in fe- male rats are lactotroph cells (Oishi et al. 1993). Although variations in the number of prolactin cells along pregnancy and lactation have been reported (Go- luboff y Ezrin 1969; Amat  and Mufioz-Barragfin 1974; Scheithauer et al. 1990), very few studies using prolifera- tion markers have been published (Kalbermann et al. 1979). Kalbermann et al. (1979) reported a decrease in BrdU incorporation in the hypophysis at the end of pregnancy, which they explained as being a result of hyperprolactine- mia. The present study - the first to use PCNA as a pro- liferation marker  - demonstrates a significant increase in proliferating lactotroph cells during late pregnancy. The discrepancies with the findings of Kalbermann could be due to the fact that PCNA is 5-fold more sensi- tive for the detection of hypophysial proliferating cells than BrdU (Oishi et al. 1993). However, our results are consistent with the massive hyperplasia of lactotroph cells described in women by Scheithauer et al. (1990) and with the increase in mitosis in lactotrophs of gestating women reported by Stefaneanu et al. (1992). In view of the elevated prolactin activity during lacta- tion - probably due to the stimulating effect of oxytocin, T R H  and VIE a decrease in dopamine tonic inhibition and suckling stimulation (Matsuzaki et al. 1997) - it is clear that during lactation lactotroph cells must be nu- merous and hyperactive, as seen here. Nevertheless, this increase in the number of cells does not agree with the finding in our study of low proliferative activity during lactation. Therefore, the need for prolactin to be pro- duced during post-partum lactation requires a previous preparative phase, which is achieved by high proliferative activity during late gestation, as demonstrated in this study. In sum, our results demonstrate that the increase in lac- totroph cells observed during lactation is a consequence of cellular proliferation in late pregnancy and, without rejecting the possibility of the appearance of mammo- somatotroph cells, suggest the existence of a preparatory proliferative phase of lactotroph cells prior to the de- mands for prolactin during lactation. Acknowledgements. Supported by the FIS 99/1187 program and the SA58/98 Social European Funds and JCyL program. References Amat R Mufioz-Barragfin L (1974) Mitosis en la adenohip6fisis. Ann Anat 23:15-27 Ben-David M, Rodbard D, Bates RW, Bridson E, Chrambach A (1973) Human prolactin in plasma, amniotic fluid and pitui- tary: identity and characterization by criteria of electrophor- esis and isoelectric focusing in polyacrylamide gel. J Clin Endocrinol Metab 36:951-964 Boockfor FR, Hoeffler JP, Frawley SL (1987) Estradiol induces a shift in cultured cells that release prolactin or growth hor- mone. Am J Physiol 250:103-106 Carbajo-P6rez E, Watanabe YG (1990) Cellular proliferation in the anterior pituitary of the rat during the postnatal period. Cell Tissue Res 261:333-338 Carretero J, Rubio M, Navarro N, Nieto E Vfizquez RJ, San- chez F, Vfizquez R (1995). In vitro modifications in the pro- liferation rate of prolactin cells are accompanied by nuclear morphometric variations. Histol Histopathol 10:135-139 Cowie AT, Forsyth IA, Hart IC (1980) Hormonal control of lac- tation. Springer, Berlin Goluboff LH, Ezrin C (1969) Effect of pregnancy on the somato- troph and the prolactin cell of the human adenohypophysis. J Clin Endocrinol Metab 29:1533-1541 Hunt TE (1943) Mitotic activity in the anterior hypophysis of fe- male rats of different age groups and at different periods of the day. Endocrinology 32:334-339 Kalbermann LE, Szijian I, Burdman JA (1979) Cell proliferation in the rat pituitary gland: A mechanism of control in prolactin cell. 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Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats

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Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats

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