Morphometric analysis of the folliculostellate cells and luteinizing hormone gonadotropic cells of the anterior pituitary of the men during the aging process

Tissue and Cell 49 (2017) 78–85

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Morphometric analysis of the folliculostellate cells and luteinizing hormone gonadotropic cells of the anterior pituitary of the men during the aging process ˇ Jovana Cukuranovi c´ Kokoris a , Ivan Jovanovic´ a,∗ , Vukica Pantovic´ b , Miljan Krstic´ c , – Ugrenovic´ a , Vesna Stojanovic´ a Milica Stanojkovic´ d , Verica Miloˇsevic´ e , Sladana a

Department of Anatomy, Faculty of Medicine, University of Niˇs, Serbia Public Health Institute Niˇs, Niˇs, Serbia c Institute of Pathology, Faculty of Medicine, University of Niˇs, Niˇs, Serbia d Institute of Forensic Medicine, Faculty of Medicine, University of Niˇs, Niˇs, Serbia e University of Belgrade, Institute for Biochemical Research “Siniˇsa Stankovi´c”, Beograd, Serbia b

a r t i c l e

i n f o

Article history: Received 3 October 2016 Received in revised form 27 October 2016 Accepted 11 November 2016 Available online 17 November 2016 Keywords: Folliculostellate cells Gonadotropes Morphometry Aging

a b s t r a c t The aim of this research was to quantify the changes in the morphology and density of the anterior pituitary folliculostellate (FS) and luteinizing hormone (LH) cells. Material was tissue of the pituitary gland of the 14 male cadavers. Tissue slices were immunohistochemically stained with monoclonal anti-LH antibody and polyclonal anti-S100 antibody for the detection of LH and FS cells, respectively. Digital images of the stained slices were afterwards morphometrically analyzed by ImageJ. Results of the morphometric analysis showed significant increase of the FS cells volume density in cases older than 70 years. Volume density of the LH cells did not significantly change, whereas their area significantly increased with age. Nucleocytoplasmic ratio of the LH cells gradually decreased and became significant after the age of 70. Finally, volume density of the FS cell significantly correlated with LH cells area and nucleocytoplasmic ratio. From all above cited, we concluded that in men, density and size of the FS cells increase with age. Long-term hypertrophy of the LH cells results in their functional decline after the age of 70. Strong correlation between FS cells and LH cells morphometric parameters might point to age-related interaction between these two cell groups. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Despite the fact that testicular morphology, semen production, and fertility express marginally changes in elderly, male gonadal axis manifests progressive testosterone deficiency during the aging process. Increased incidence of sarcopenia, osteoporosis, cardiovascular disorders, insulin resistance, disorders of cognitive function and, generally impaired quality of life of aging men are possible consequences of such age-related decline (Bhasin et al., 2000; Mulligan et al., 1999; van Beld and Lamberts, 2002; Veldhuis, 2008). Biochemically, older men are characterized by more frequent peaks of serum luteinizing hormone (LH), reduced serum LH peak amplitudes, lower concentrations of total and especially bioavailable

∗ Corresponding author at: Department of Anatomy, Faculty of Medicine, Blvd. Dr. Zoran Djindjic´ 81, 18000 Niˇs, Serbia. ´ E-mail address: [email protected] (I. Jovanovic). http://dx.doi.org/10.1016/j.tice.2016.11.006 0040-8166/© 2016 Elsevier Ltd. All rights reserved.

(free) testosterone, as well as greater disorderliness of LH and testosterone release (Bhasin et al., 2000; Mulligan et al., 1999; Veldhuis, 2013). Previous studies pointed out that primary mechanisms mediating gradual testosterone depletion in older men are still unknown. Beside the effects of medications, comorbidity and intercurrent illness, age-related decline in serum testosterone concentrations is predominantly a consequence of decreased production, because its plasma clearance rates are, in fact, lower in older than in younger men (Bhasin et al., 2000). Decline of the testosterone production could be caused by disrupted function of any of the three key control sites within the male reproductive axis, namely, the hypothalamus, anterior pituitary, or Leydig cells of the testes. Majority of the investigators were focused on the aging changes of suprapituitary structures and, age-related changes at the level of the testicular tissue. At suprapituitary level, ultrastructural changes of the gonadotropin releasing hormone (GnRH) neurons are considered responsible for the attenuation of the endogenous GnRH and

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consequent smaller spontaneous LH pulses in the elderly men. Further, attenuated ability of LH to stimulate testicular testosterone secretion, qualitative difference in the LH isoform profiles in older males, impaired Leydig cell perfusion, decline of the total Leydig cell volume and the absolute number, fusion of Leydig cells with the formation of the multinucleated cells, which functionality is not known, or a combination of these disruptions could cause impaired testicular steroidogenesis in aging men (Bhasin et al., 2000; Mulligan et al., 1999; Strauss et al., 2009). According to the literature data, aging is not associated with measurable impair of gonadotropes’ secretion of either biologically active or immunologically reactive LH (Mulligan et al., 1999). However, instead of unleashing LH secretion by withdrawing negative feedback, lower testosterone concentrations observed in elderly men are generally associated with preserved, in some cases even elevated, but more irregular LH secretion (Veldhuis et al., 2007). Nevertheless, causes of such progressive irregularity of the LH and consequent testosterone release with age remain vague. Impairment of the efficacy of the testosterone feedback due to decline of the androgen-receptor expression in the brain and pituitary gland in the older men could represent one possible cause (Veldhuis et al., 2007; Veldhuis, 2008). But the simultaneous presence of other factors that can, beside GnRH, additionally directly stimulate synthesis and secretion of the LH at the pituitary level, cannot be excluded. In their essay Schwartz and associates cited that the function of the adenohypophysis probably represents the result of the integration of the multiple signals received, including hypothalamic, peripheral and intrapituitary (SCHWARTZ et al., 1998). Intrapituitary factors may exhibit either stimulatory or inhibitory effects on the adenohypophyseal hormones production. Thus, production and release of LH is, also, under the control of various locally produced signaling molecules that form a complex network and participate in an autocrine/paracrine control of gonadotropes function (PerezCastro et al., 2012). According to Denef gonadotropes are involved in cross-talk with both hormonal, mainly lactotropes, somatotropes and corticotropes, and with nonhormonal cells (Denef, 2008). Folliculostellate (FS) cells are non-hormonal cells which comprise 5–10% of the anterior pituitary’s cells population. Morphology of these cells strongly suggests that they have a role in the microcirculation of the nutrients, ions and waste products. They form a communication system within the anterior pituitary, which coordinates the release of hormones from its different parts. Also, FS cells can release many substances into the intercellular matrix, which can influence the function of the neighboring hormonal cells in a paracrine manner (Morris, 2011; Renner et al., 2009). Internal milieu of the elderly men changes with age (Deleidi et al., 2015; Franceschi et al., 2000) and we assume that such changes can stimulate FS cells interactions with gonadotropes via their paracrine loops. These interactions could consequently alter the function of the gonadotropes, which would finally result in age-related alterations of plasma LH levels. Taking into the consideration the fact that histopathological studies about the gonadotropes’ aging changes are limited, the aim of our research was to morphometrically analyze the changes in the morphology and density of the anterior pituitary’s FS cells and LH gonadotropic cells, and to subsequently statistically establish possible connection between such changes, thereby indirectly associating them with the possible morphological substrate that underpins LH aging changes.

2. Material and methods Material was tissue of the pituitary gland of the 14 male cadavers which age ranged from 41 to 87 years. Tissue samples were, in accordance with the rules of the Internal Ethics Committee, obtained during the routine autopsies performed at the

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Department of the forensic medicine at Faculty of Medicine in Niˇs, Serbia, with post mortem period being no longer than 24 h. Cadavers used in this study have been ante mortem without any previously diagnosed neurological, psychiatric or endocrine disorder. Visible brain or pituitary damages were not observed during the autopsies. Also, pathohistological assessment of the brain and pituitary gland excluded the presence of possible latent or misdiagnosed disease. Cadavers were, according to their age, classified into three age groups: first (I), which included the cases old from 30 to 49 years, second (II), with the cases 50–69 years old, and the third with the cases old 70 years and older. Obtained pituitaries were further fixed for 24 h in the 10% buffered formalin and afterwards cut by transversal section into the dorsal and ventral half, which were finally embedded in paraffin. Thereafter, tissue sections 5 ␮m thick were made for both dorsal and ventral half of the hypophysis, stained routinely with H&E and then immunohistochemically processed. Immunohistochemical analysis included staining of the tissue slices with monoclonal rat anti-LH antibody (kindly provided by Dr Parlow-a, NIH, Bethesda, Md., USA; PAP kit; 1:100), for the detection of the LH gonadotropic cells (Medigovic´ et al., 2012), and polyclonal rabbit anti-S100 antibody (Dako, polyclonal rabbit anti-cow S-100, code no. Z0311; Streptavidin HRP, Novocastra Peroxidase Detection System, Novocastra, UK; 1:400), for the detection of the FS cells. Stained slices were histologically analyzed with light microscope under the 4× and 40× lens magnification. Morphometric analysis was performed on digital images captured with digital camera (1.3 megapixel) under the 40× lens magnification. Thirty fields of vision of the anterior pituitary were captured from both the slices of its dorsal and ventral half, totally 60 fields of vision per each analyzed case. Ten fields of vision were acquired from each of the anterior pituitary’s lateral wings, and 10 from the glands mucoid wedge of both dorsal and ventral halves of each analyzed case (or totally 40 fields of vision from the adenohypophyseal lateral wings, and 20 from its central wedge, per each analyzed case). Image analysis was performed by ImageJ (https:// imagej.nih.gov/ij/). In case of LH cells, planimetric analysis included the measurement of their area (ALH ) and nuclear area (ANLH ). The calculation of the LH cells nucleocytoplasmic ratio ((N/C)LH ) was obtained as a ratio between the nuclear and cytoplasmic area, which was obtained as difference between the total area of the cell and area of its nucleus. Sixty randomly selected LH cells were measured per one slice of both dorsal and ventral half of the anterior pituitary in each case (totally 120 cells per one case). Stereological analysis was performed with multipurpose M168 stereological grid (d = 17.88 ␮m, a = 15.49 ␮m2 , AT = 2601.54 ␮m2 , LT = 1501.92 ␮m), which was overlaid over the analyzed digital images of the histological slices. Volume density of the LH cells (VVLH ), and FS cells (VVFSC ) was obtained as a ratio of the number of points of the grid which hit the immunopositive cells (PF ) and the total number of the points of the grid (PT = 168), for each of the analyzed fields of vision of the dorsal and ventral half of the hypophysis (Russ and Dehoff, 2000). Values of the area, nuclear area, nucleocytoplasmic ratio and volume density of LH cells, and volume density of the FS cells for each analyzed case was obtained as average of the values of all measured fields of vision, respectively. Statistical analysis was performed by SPSS statistical package (version 16). Correlation between the age and measured morphometric parameters, as well as between the morphometric parameters of different cell types, was assessed by linear correlation and linear regression analysis. More detailed estimation of the dynamics of the morphometric parameters of the measured cell types with age was analyzed by One Way ANOVA and Tukey Kramer multiple comparison test. Due to small size of the analyzed sample, obtained statistically significant differences were additionally

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verified by the calculation of the corresponding effect sizes (Lipsey et al., 2001). 3. Results 3.1. Histological analysis Fig. 1A presents LH cells of the anterior pituitary of the 41 years old case of the first group. In these cases, LH cells were scattered across the lateral wings of the anterior pituitary, whereas in the mucoid wedge of the gland these cells were mainly localized inside acinar formations which number varied in different cases. Gonadotropic LH cells of such cases were oval or polygonal and with centrally, or more frequently eccentrically located immunonegative blue stained euchromatic nucleus (Fig. 1A). Fig. 1B presents pars distalis of the case of the second group, 65 years old. In this group, increase of the size of the LH cells can be observed, whereas their distribution and density, as well as morphology of their nuclei remained similar to the same of the first group cases. Finally, Fig. 1C presents anterior pituitary of the case 87 years old, of the third group. Similar to the second group (Fig. 1B), these cases were characterized with various degree of the interstitial fibrosis and the reduction of the size of the blood vessels’ lumina. Distribution and density of the LH cells were not significantly different from the same of both second and the first group cases. However, beside further increase of the size of these cells, their oval shape, smaller, eccentrically located, hyperchromatic nuclei were more frequent in relation to the cases of the above described two groups. Immunohistochemical reaction of the cytoplasm of the LH cells was positive and similar in all three analyzed groups. It was brown stained with emphasized granular pattern due to the presence of numerous secretory granules (Fig. 1A–C). Folliculostellate cells of the anterior pituitary of the first group cases were characterized with star-like appearance, immunopositive cell body and slender processes which extended between the endocrine cells. They were sparse and irregularly distributed in the lateral wings, as well as, mucoid wedge of the gland (Fig. 1D). In the mucoid wedge, FS cell bodies, or their extensions were, together with the endocrine cells, observed inside the walls of the follicular formations. In the second group, density of the FS cells did not significantly change in relation to the first group cases. Though, irregularity of the shape of the FS cell bodies made evaluation of their size difficult during our histological analysis, its results pointed to the conclusion that in this group their cell bodies became generally larger in relation to the same of the first group cases (Fig. 1E). Finally, in the third and the oldest group, significant increase of the FS cells number in both intermediate part and lateral wings of the adenohypophysis was observed in addition to the further increase of their size (Fig. 1F).

nucleocytoplasmic ratio of the LH cells, as well as volume density of the FS cells (Table 2). Thus, age explained 90% of the variance of the area and 86% of the variance of the nucleocytoplasmic ratio of the LH cells, and 52% of the variance of the volume density of the FS cells of the anterior pituitary. This, in all later cited cases, represented a large effect size and could be identified by the models given in the Table 2. More detailed dynamics of the LH cells and FS cells morphometric parameters with age was assessed by One Way ANOVA test. Its results showed that the average area and nucleocytoplasmic ratio of LH cells significantly differed between analyzed age groups (Table 3). These differences were characterized with large effect size (ALH : d = 0.93; (N/C)LH : d = 0.93). Average area of the LH cells of the second group cases was significantly higher than the one of the first group cases. Mean area of the latter cells of the third group cases was significantly higher than the same of the second and the first age group cases (Table 3, Fig. 2D). However, in case of their nucleocytoplasmic ratio, mean value of the third age group was significantly lower than the ones of the second and the first age group, while its value in the second age group was lower than the first, but this difference was not significant (p > 0.05) (Table 3, Fig. 2E). Finally, mean volume density of the FS cells significantly differed between the analyzed age groups, and these differences were characterized with the large effect size (d = 0.96) (Table 3). Similarly to the nucleocytoplasmic ratio of the LH cells, average volume density of the FS cells of the third age group was significantly higher than the ones of the first age group (Table 3, Fig. 2F), while mean values of this parameter were higher in the second in relation to the first, and in the third in relation to the second age group, but these differences were insignificant (p > 0.05). All other analyzed morphometric parameters of the measured cell types did not significantly differ between the evaluated age groups (p > 0.05). Results of the correlation analysis between the morphometric parameters of the LH cells and FS cells showed that volume density of the FS cells significantly positively correlates with the area of the LH cells (R = 0.68, p = 0.007, N = 14) (Fig. 2G). Opposite to the latter, volume density of the FS cells significantly negatively correlated with the nucleocytoplasmic ratio of the LH cells (R = −0.59, p = 0.026, N = 14) (Fig. 2H). Linear regression analysis additionally confirmed that volume density of the FS cells significantly predicts area (F(1,12) = 10.33, p = 0.007) and nucleocytoplasmic ratio (F(1,12) = 6.42, p = 0.03) of the LH cells. These relationships can be identified by the following models: ALH = 109.42 + VVFSC × 17.25 and (N/C)LH = 0.271 − VVFSC × 0.025, which explained 42% of the variance of the area (adjusted R2 = 0.42) and 29% of the variance of the nucleocytoplasmic ratio (adjusted R2 = 0.29) of the LH cells. All latter described relationships were characterized with the large effect sizes.

3.2. Morphometric analysis

4. Discussion

Results of the morphometric analysis of the LH cells and FS cells of all analyzed cases are presented in Table 1. Correlation analysis between the age and the morphometric parameters of the LH cells showed that their area significantly increases (R = 0.95; p < 0.001; N = 14) (Fig. 2A), whereas their nucleocytoplasmic ratio significantly decreases with age (R = −0.86; p < 0.001; N = 14) (Fig. 2B). Volume density of the FS cells significantly increased during the aging process, too (R = 0.75; p = 0.002; N = 14) (Fig. 2C). Nuclear area of the LH cells did not significantly change with age (p > 0.05), while their volume density increased during the aging, but this increase was not significant (p > 0.05). Results of the bivariate linear regression analysis additionally showed that the age significantly predicts area and

As far as the dynamics of the FS cells with age is concerned, the results of the morphometric studies gave no straight answer. However, the majority of them pointed to the postnatal increase of their number. Thus, Soji et al. observed that the population of the rat FS cells increases postnatally until day 40, after which the levels plateau (Soji et al., 1994). Afterwards, synchronous postnatal increase of the FS cell number in male Wistar rats’ anterior pituitaries was reported by Allaerts and associates (Allaerts et al., 1997). Opposite to the latter two research groups, Console and colleagues found a progressive age-related decline in the number of the FS cells in both male and female rats (Console et al., 2000). Finally, the results of the morphometric study by Pavlovic and colleagues, performed on men and women cadaveric anterior pituitaries, showed

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Fig. 1. Adenohypophyseal gonadotropes: (A) tissue section of a 41 year old male case; immunopositive LH cells with eccentric or centrally located euchromatic nucleus (arrows); (B) tissue section of a 65 year old male case; larger immunopositive LH cells with eccentrically or centrally located euchromatic nucleus (arrows); (C) tissue section of the 87 year old male case; large immunopositive LH cells with small eccentrically located hyperchromatic nucleus (arrows); anti-LH antibody; PAP; 40× lens magnification; Folliculostellate cells of the adenohypophysis: (D) tissue section of the 45 years old male case; S100 immunopositive FS cells with immunopositive nucleus inside the cell body and slender processes which extend from the cell body between the immunonegative endocrine cells (arrows); (E) tissue section of the 65 year old male case with larger S100 immunopositive FS cells (arrows); (F) tissue section of the 87 years old male case; large and more numerous FS cells (arrows); anti-S100 antibody; Novocastra Peroxidase Detection System; 40× lens magnification.

Table 1 Results of the morphometric analysis of the LH cells and FS cells of the anterior pituitary of all 14 analyzed cases. Case

Age

Group

ALH (mm2 )

ANLH (␮m2 )

(N/C)LH

VVLH (%)

VVFSC (%)

3 5 2 4 8 9 6 7 10 11 12 13 15 14

41 45 48 48 57 61 65 65 66 76 76 77 78 87

I I I I II II II II II III III III III III

120.82 124.03 115.74 123.45 129.22 134.94 151.48 140.77 153.08 184.42 160.11 169.88 174.97 194.00

28.09 24.31 22.48 24.47 22.58 26.93 27.80 22.87 29.43 27.90 19.99 27.37 25.52 28.32

0.30 0.24 0.24 0.25 0.21 0.25 0.23 0.19 0.24 0.18 0.14 0.19 0.17 0.17

3.04 4.03 3.57 3.68 4.73 8.39 4.99 2.71 9.24 6.48 3.15 5.42 3.07 5.19

1.13 1.00 1.53 1.40 1.20 2.75 3.96 2.10 1.88 1.91 3.22 2.44 3.34 3.74

the aging-related increase of the density of FS cells, which became significant in the group of cases older than 80 years (Pavlovic´ et al.,

2013). Results of our study are mainly in agreement with the latter cited study (Pavlovic´ et al., 2013) and study by Allaerts and

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Fig. 2. (A) Correlation between the age and area of LH cells of the anterior pituitary; (B) correlation between the age and nucleocytoplasmic ratio of the LH cells of the pars distalis of the adenohypophysis; (C) correlation between the age and volume density of the anterior pituitary’s FS cells; (D) mean area of the adenohypophyseal LH cells of the analyzed age groups; (E) mean nuclecytoplasmic ratio of the pars distalis LH cells; (F) mean volume density of the anterior pituitary’s FS cells of the evaluated age groups; (G) Correlation between the volume density of the FS cells and area of the LH cells; (H) Correlation between the volume density of the FS cells and nucleocytoplasmic ratio of the LH cells; a − I: II, p < 0.05; b − I: III, p < 0.05; c − II: III, p < 0.05.

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Table 2 Regression analysis between the age as predictor and area, nucleocytoplasmic ratio of the LH cells, as well as volume density of the FS cells as outcome variables. ALH ␤

Variable B SEB 40.91 10.37 Constant 1.69 0.16 Age R2 = 0.90; F(1,12) = 112.36, p < 0.001; Model: ALH = 40.91 + Age × 1.69

0.95

t 3.94 10.6

p 0.002 < 0.001

t 13.51 −5.94

p < 0.001 < 0.001

t −1.24 3.90

p 0.24 0.002

(N/C)LH ␤

Variable B SEB 0.376 0.028 Constant −0.0025 0.0004 Age R2 = 0.86; F(1,12) = 32.29, p < 0.001; Model: DFLH = 0.376 − Age × 0.0025

−0.864

VVFSC ␤

Variable B SEB −1.10 0.88 Constant 0.05 0.01 Age R2 = 0.52; F(1,12) = 15.21, p = 0.002; Model: VVFSH = Age × 0.05 − 1.10

0.75

Table 3 Descriptive statistics of the analyzed morphometric parameters of the LH cells and FS cells; results of One Way ANOVA test of the latter cited parameters between the analyzed age groups. Variable

Group

N

Mean

SD

SE

95% CI

Tukey-Kramer post hoc test

LB

UB

I 4 II 5 III 5 F(2,11) = 34.37, p < 0.001

121.01 141.90 176.68

3.78 10.34 13.07

1.89 4.62 5.85

115.00 129.06 160.45

127.03 154.73 192.91

a,b a,c b,c

I 4 II 5 III 5 F(2,11) = 0.17, p = 0.85

24.84 25.92 25.82

2.35 3.05 3.43

1.18 1.37 1.53

21.10 22.13 21.56

28.58 29.71 30.08

/ / /

I 4 5 II III 5 F(2,11) = 17.78, p < 0.001

0.26 0.22 0.17

0.03 0.02 0.02

0.01 0.01 0.01

0.22 0.20 0.15

0.30 0.25 0.19

b c b,c

4 I II 5 III 5 F(2,11) = 1.88, p = 0.20

3.58 6.01 4.66

0.41 2.72 1.50

0.21 1.22 0.67

2.93 2.63 2.80

4.23 9.39 6.52

/ / /

4 1.26 I 5 2.43 II 5 2.93 III F(2,11) = 5.01, p = 0.03 ANOVA a − I: II, p < 0.05; b − I: III, p < 0.05; c − II: III, p < 0.05

0.24 1.07 0.74

0.12 0.48 0.33

0.88 1.10 2.01

1.65 3.76 3.85

b / b

ALH (␮m2 )

ANOVA ANLH (␮m2 )

ANOVA (N/C)LH

ANOVA VVLH (%)

ANOVA VVFSC (%)

associates (Allaerts et al., 1997). However, beside the fact that male cases studied by Pavlovic´ and associates (Pavlovic´ et al., 2013) were older and characterized by higher values of FS cells volume density than ours, latter cited authors (Pavlovic´ et al., 2013) explained observed significant increase of the FS cells volume density by the simultaneous increase of these cells size and number in the mucoid wedge of the adenohypophysis in their oldest group. Our histological analysis did not reveal any regional differences in the dynamics of the FS cells during the aging of the men. Also, its results led us to assumption that aging-related increase of the FS cells volume density, detected by our research group, represents a two-staged process. The first phase of such process, which probably occurs in the men 50–70 years old, could predominantly be the consequence of the increase of the FS cells size, or their hypertrophy, while in the second phase, which takes place in the men older than 70 years, further increase of the FS cells size and simultaneous increase of their number occur, causing in such way significant increase of their volume density. So, ultimately, majority of the latter cited facts could indirectly indicate possible elevation of the FS cells function in aging men.

Morphometric studies about the gonadotropes aging changes are infrequent, too. Opposite to the other hormonal cells of the adenohypophysis, which store one hormone in separate cell types, many gonadotropes show immunoreactivity for both LH and FSH. This additionally makes their quantification with age more difficult, because, in such cells, expression of FSH and LH may be quite different in different phases of the postnatal period. Meeran et al. evaluated the changes of the subtypes of gonadotropes in pubertal and adult rhesus monkeys and concluded that number of gonadotropes increases in the adults in relation to the juvenile rhesus monkeys, predominantly due to increase of the number of LH, as well as bihormonal cells (Meeran et al., 2003). Console et al. performed morphometric analysis of the gonadotropes in young, old and senescent rats and, established a progressive age-related reduction in the cellular density, volume density and surface density of LH cells. On the other hand, perimeter and area of the gonadotropes showed increase in young and old, but appeared drastically reduced in senescent rats. Basal serum levels of LH and FSH showed the same trend as area and perimeter of the gonadotropes (Cónsole et al., 1994). Kurosumi et al. classified

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gonadotropes of the rats according to the size of their secretory granules into two types: type I which contained large and small secretory granules and expressed both FSH and LH, and type II which contained only small granules and were immunopositive on LH. They found that in young adult male rats, type I gonadotropes are more frequent than type II. In middle-aged rats type I gonadotropes were still predominant, but most of them poorly expressed FSH. In old-aged male rats type I to II ratio became reversed when compared with that at young and middle aged, with type II gonadotropes much more commonly observed. Kurosumi et al. finally concluded that, as the age of the rat advances, LH cells become predominant in the male pituitary, which is accompanied by remarkable decrease of FSH and very slow decrease of LH content (Kurosumi et al., 1991). In our case, the density of the LH cells did not significantly change with age. However, differently from the Console and associates, area of the LH cells significantly increased, even after the age of 70 years. Opposite to the area, nucleocytoplasmic ratio gradually decreased with age, and this decrease became significant after the age of 70, similarly to the volume density of the FS cells. So, our findings of relatively stable density of the LH cells population and the increase of their size with age, together with previously reported by some authors age-related elevation of the LH production (van Beld and Lamberts, 2002), led us to the conclusion that latter cited cells probably develop hypertrophy (Mitchell et al., 2007) in men during the aging. This might indicate their exposure to increased functional stress with time, probably due to impaired feedback by testosterone, or overstimulation by some extra-, or intrapituitary factors. Gradual decline of the LH cells nucleocytoplasmic ratio represents the sign of their functional decline, which becomes significant after the age of 70, the most likely because of their exhaustion caused by long-term hypertrophy. This is in agreement with the observed slow decline of their LH content by Kurosumi and associates (Kurosumi et al., 1991). Nevertheless, with the exception of the higher irregularity and smaller amplitude of the LH pulses, as we described above, in the scientific community predominant position is that the secretion of LH not only does not notably change, but that it could be said, it mildly increases with age in men (Bhasin et al., 2000; Mulligan et al., 1999; Strauss et al., 2009). This is in opposition with the finding of diminished secretion of GnRH, as well as with the assumption that impaired testosterone feedback at hypothalamic and pituitary level unleashes LH secretion during the aging (Veldhuis et al., 2007; Veldhuis, 2008). The fact that FS cells have processes that extend to gonadotropes (Denef, 2008), then, significant correlation between the FS cells volume density and LH cells area and nucleocytoplasmic ratio in our case, suggests a particular relationship between these two anterior pituitary cell types and could point to their increased aging-related interaction. But the question about the possible mechanism(s) which underlies, as well as its nature, remains to be seen. General alterations in endocrine homeostasis with age may be associated with the changes in the intrapituitary factors as local adaptive response (Perez-Castro et al., 2012). Folliculostellate cells are members of the dendritic cell meshwork of the human body, contain receptors for many cytokines and probably represent the targets for the pro-inflammatory cytokines increased in elderly men, such as TNF-␣ and IL-1 (Deleidi et al., 2015; Denef, 2008; Franceschi et al., 2000; Minciullo et al., 2016; Ray and Melmed, 1997). Further, latter cited cytokines may stimulate FS cells to produce and release different paracrine agents, such as IL-6 (Ray and Melmed, 1997; Renner et al., 2009; Yamaguchi et al., 1990), follistatin (Aroua et al., 2012; Bilezikjian et al., 2006, 2003; Coss et al., 2005; Denef, 2008; Morris, 2011; Perez-Castro et al., 2012), glutamine (Denef, 2008), IGF-1 (Denef, 1986; Schwartz, 2000; Winters and Moore, 2004) and NO (Denef, 2008; Gomariz et al., 2006;

McCann et al., 2005; Perez-Castro et al., 2012) which can, in the gonadotropes of the elderly men, set the balance between LH and FSH synthesis and secretion in favor of LH, as Kurosumi and colleagues suggested (Kurosumi et al., 1991). Finally, from all above cited facts, we concluded that in men, there is increase of density, size and, probably function of the FS cells of the anterior pituitary with age. Despite, in the literature already established impaired secretion of hypothalamic GnRH, LH cells becomes hypertrophic during the aging process and, their long-term hypertrophy probably results in their eventual functional decline after the age of 70. Strong correlation between FS cells and LH cells morphometric aging changes might point to increasing interaction between these two groups of cells, probably due to FS cells paracrine activity in aging men. For the verification of the altered paracrine interactions of the FS and LH cells with age, future studies should be focused on the evaluation of the topographical arrangement of the LH cells and FS cells and simultaneous detection of the presence of the local paracrine factors by double staining immunohistochemistry techniques in the pituitaries of the younger control and healthy elderly individuals. Direct effects of these paracrine substances on LH release during the aging process should be established in vitro by their application in the culture of the LH cells derived from younger control and healthy elderly experimental animals. Conflict of interest None. Acknowledgement This work was supported by Grant No. 175092 from the Ministry of Education and Science and Technical Development of Serbia. References Allaerts, W., Salomon, B., Leenen, P.J., van Wijngaardt, S., Jeucken, P.H., Ruuls, S., Klatzmann, D., Drexhage, H.A., 1997. A population of interstitial cells in the anterior pituitary with a hematopoietic origin and a rapid turnover: a relationship with folliculo-stellate cells? J. Neuroimmunol. 78, 184–197. Aroua, S., Maugars, G., Jeng, S.-R., Chang, C.-F., Weltzien, F.-A., Rousseau, K., Dufour, S., 2012. Pituitary gonadotropins FSH and LH are oppositely regulated by the activin/follistatin system in a basal teleost, the eel. Gen. Comp. Endocrinol. 175, 82–91, http://dx.doi.org/10.1016/j.ygcen.2011.10.002. Bhasin, S., Huang, G., Travison, T.G., Basaria, S., 2000. Age-Related Changes in the Male Reproductive Axis. 2014 Feb 14. In: De Groot, L.J., Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch, C., Korbonits, M., McLachlan, R., New, M., Purnell, J., Rebar, R., Singer, F., Vinik, A. (Eds.), Endotext [Internet]. MDText.com, Inc, South Dartmouth (MA), Available from http:// www.ncbi.nlm.nih.gov/books/NBK278998/, PubMed PMID: 25905229. Bilezikjian, L.M., Leal, A.M.O., Blount, A.L., Corrigan, A.Z., Turnbull, A.V., Vale, W.W., 2003. Rat anterior pituitary folliculostellate cells are targets of interleukin-1␤ and a major source of intrapituitary follistatin. Endocrinology 144, 732–740, http://dx.doi.org/10.1210/en.2002-220703. Bilezikjian, L.M., Blount, A.L., Donaldson, C.J., Vale, W.W., 2006. Pituitary actions of ligands of the TGF-␤ family: activins and inhibins. Reproduction, http://dx.doi. org/10.1530/rep.1.01073. Cónsole, G.M., Gómez Dumm, C.L., Goya, R.G., 1994. Immunohistochemical and radioimmunological study of pituitary gonadotrophs during aging in male rats. Mech. Ageing Dev. 73, 87–95. Console, G.M., Jurado, S.B., Riccillo, F.L., Gomez Dumm, C.L., 2000. Immunohistochemical and ultrastructural study of pituitary folliculostellate cells during aging in rats. Cells Tissues Organs 167, 25–32, http://dx.doi.org/10. 1159/000016763. Coss, D., Thackray, V.G., Deng, C.-X., Mellon, P.L., 2005. Activin regulates luteinizing hormone beta-subunit gene expression through Smad-binding and homeobox elements. Mol. Endocrinol. 19, 2610–2623, http://dx.doi.org/10.1210/me. 2005-0047. Deleidi, M., Jäggle, M., Rubino, G., 2015. Immune ageing, dysmetabolism and inflammation in neurological diseases. Front. Neurosci. 9, http://dx.doi.org/10. 3389/fnins.2015.00172. Denef, C., 1986. 1 Paracrine interactions in the anterior pituitary. Clin. Endocrinol. Metab. 15, 1–32, http://dx.doi.org/10.1016/S0300-595X(86)80040-8. Denef, C., 2008. Paracrinicity: the story of 30 years of cellular pituitary crosstalk. J. Neuroendocrinol, http://dx.doi.org/10.1111/j.1365-2826.2007.01616.x.

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