Effects of somatostatins on gonadotrophic cells in female rats

Acta histochem. 100,329-335 (1998) © Gustav Fischer Verlag

Effects of somatostatins on gonadotrophic cells in female rats Mirjana Lovren, Milka Sekulic, Verica Milosevic and Natasa Radulovic Institute for Biological Research "Sinisa Stankovic", 29 Novembra 142, YU-l1060 Belgrade, Yugoslavia Accepted 17 May 1998

Summary The effects of intracerebroventricular application of SRIF-14 and SRIF-28 on pituitary gonadotrophic cells (FSH and LH) were examined using immunocytochemical and morphometrical methods in adult female Wistar rats. FSH- and LH-producing cells were studied using the peroxidase-antiperoxidase (PAP) immunohistochemical procedure. Morphometry and stereology were used to evaluate changes in the number, volume densities and relative volume densities of LH- and FSH-immunopositive cells. In females treated with SRIF-14 or SRIF-28, the gonadotrophs were smaller, often pycnotic and more intensely stained. The number of LH-positive cells per unit area (mm2 ) was significantly decreased in both somatostatin-treated groups, while the number of FSH-positive cells was similar to that in the controls. Volume densities of perykarya of LH- and FSH-positive cells were decreased in all treated females, but extremely different in LH-positive cells after SRIF-14 administration. The relative volume density of LH cells was significantly decreased in both somatostatin-treated groups, while immunopositive FSH cells were not significantly decreased compared with the controls. It can be concluded that centrally applied somatostatins lead to changes in the immunocytochemical and morphometric properties of LH cells, while there is no significant effect on FSH cells. Key words: somatostatin - SRIF-14 - SRIF-28 - gonadotrophic cells - immunohistochemistry - stereology

Correspondence to: M. Lovren

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Introduction Somatostatin was originally detected by Krulich et ai. (1968) during purification of growth hormone releasing factor (GHRH) from sheep hypothalami. In 1973 Brazeau et ai. isolated a tetradecapeptide from bovine hypothalamus which inhibited the release of growth hormone and which was named somatostatin or SRIF-14 (somatostatin release inhibiting factor). A second peptide consisting of 28 amino acids with the complete sequence of somatostatin-14 at the carboxy terminus was subsequently discovered in the gut (Pradayrol et ai. 1980) and hypothalamus and was named SRIF-28 (Schally et al. 1980). The portal blood contains both SRIF-14 and SRIF-28 in a 70/30 ratio, which indicates that both peptides are synthesized in neuroendocrine hypothalamic neurones and are released from the terminals of the median eminence. These neuropeptides act as both hormones and neurotransmitters and possess similar biological activity (Epelbaum and Bertherat, 1993). SRIF affects cognitive and behavioural processes, the endocrine, gastrointestinal and cardiovascular systems and has also tumor growth inhibiting effects. In the pituitary gland, SRIF dramatically inhibits the release of GH from somatotrophs and blocks the release of thyroid stimulating hormone (TSH), prolactin and adrenocorticotropic hormone (Lamberts, 1988). Several studies indicate that somatostatins do not affect gonadotrophin release (Brazeau et aI., 1973; Reichlin, 1983). However, McCann (1982) hypothesized that sufficiently large somatostatin doses could block the release of all pituitary hormones. This prompted us to examine whether intracerebroventricular administration of SRIF-14 or SRIF-28 influences immunocytochemical and mophometric characteristics of gonadotrophic cells in adult female rats.

Material and Methods Animals. Virgin female Wistar rats (60 days old, body weight 240 ± 10 g) maintained at 25 ± 2 °C with a 12/12 h light/dark cycle and free access to water and food were used. Surgical procedures were performed under ether anesthesia. A headset was implanted into the rats and used for intracerebroventricular (ICV) injections. A minimum recovery time of five days was allowed before the experiments were started. The headset consisted of a Silastic-sealed 20-gauge cannula (Starcevic et aI., 1988), implanted into a lateral cerebral ventricle, 1.0 mID posterior and 1.5 mID lateral to the bregma and 3 mID below the cortical surface. A small stainless cerebroventricular steel cannula and a screw were cemented in the skull with dental acrylic resin (Sigmal; ICN Galenika). Treatment protocols. After recovery from surgery, the rats were divided into three experimental groups each consisting of five animals. The rats of the first and the second group received ICV three 1.0 Jlg doses of SRIF-14 or SRIF-28 (S 9129 and S 6135; Sigma, St Louis, Mo., USA), respectively, every second day. The third group served as the control and received physiological saline. All animals were killed by decapitation under deep ether anesthesia five days after the last injection.

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Immunohistochemistry. The pituitaries were removed, weighed, fixed in Bouin's solution and embedded in paraffin. For immunocytochemistry, series of seven 5.0 !lm thick sections cut through three tissue levels (dorsal, middle and ventral portion) of the pars distalis were used. The pituitary gonadotrophs were localized by a peroxidase-antiperoxidase (PAP) method. Reduction of nonspecific background staining was achieved with non-immune porcine serum (1: 100; 45 min). Anti-rat P-LH serum (diluted with PBS 1: 200) and anti-rat P-FSH serum (diluted with PBS 1: 200) for 60 min at room temperature served as the primary antibodies (a generous gift from Dr A. F. Parlow, National Institute of Health, Bethesda, MD, USA). After incubation with the second antibody (swine-antirabbit IgG, 1: 100; 45 min) the sections were tryated with rabbit peroxidase-antiperoxidase serum (1 : 100; 45 min; Dako A/S, Glostrup, Denmark). Visualization was performed with TRIS-HCI buffered saline, pH 7.4 containing 3,3'-diaminobenzidine tetrahydrochloride (DAB, Serva, Heidelberg, Germany) followed by counterstaining with haematoxylin. Control sections were incubated without primary antisera. Morphometry. FSH-P and LH-P immunoreactive cells were stereologically analyzed by simple point counting (Weibel, 1979). Morphometric parameters (the mean volume of the FSH and LH cell cytoplasm as well as the number of these immunoreactive cells per mm 2 ) were measured exactly as described previously (Lovren et al., 1996). The relative volume densities of FSH- and LH-positive cells were given as percentages of total pituitary cells in mm 3 . The data obtained for all rats of each group were combined. The means and standard errors of the mean (SEM) were calculated and statistically evaluated using Student's t-test.

Results Intracerebroventricular administration of both types of somatostatin to female rats led to an insignificant decrease of body weight as well as of absolute and relative pituitary mass when compared to the controls (Table 1). In controls, the ovoid or polyhedral FSH and LH immunoreactive cells were present occasionally alone or frequently in clusters throughout the pars distalis and in close contact with blood capillaries. After SRIF-14 or SRIF-28 treatment, gonadotrophic cells were smaller, often pycnotic and more intensely stained (Figs. 1 a, b). In SRIF-14-treated females, the number of FSH-positive cells per unit area (mm 2 ) was similar to that of controls, while the number of LH-positive cells was significantly decreased (by 17%). In animals treated with SRIF-28, Table 1. Effects of intracerebroventricularly administered SRIF-14 or SRIF-28 on body weight and absolute and relative weights of the anterior pituitary gland in female rats (mean ± SEM; n = 7) Treatment

body weight (g)

Absolute pituitary mass (mg)

Relative pituitary mass (mg%)

Control SRIF-14 SRIF-28

238.0 ± 6.4 234.0±4.0 220.0 ± 12.2

10.5 ± 0.4 9.2 ±0.6 10.0 ± 0.4

4.3 ± 0.1 3.9 ± 0.23 4.1 ± 0.4

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Fig. 1. Pituitary immunoreactive LH cells in rat females. 1a. Control rats; 1b. Fe-

males ICV-treated with SRIF-14. Arrows indicate small pycnotic LH-positive cells. x 608

the number of LH-positive cells per unit area was significantly decreased (by 25%) and that of FSH-positive cells by 5% in comparison with the controls (Fig. 2 a). Stereological analyses revealed a significantly decreased relative volume density of LH cells after administration of both somatostatins (9% and 11% after SRIF-14 and SRIF-28, respectively); in the control group the relative volume density was 19%. Concerning immunopositive FSH cells this parameter in somatostatin-treated females did not differ significantly from the controls (Fig. 2 b). Volume densities of perykarya of FSH- and LH-positive cells were lower in all treated females than in controls. Extremely different volume densities were observed in LH-positive cells after SRIF-14 administration (55% lower) (Fig.2c).

Discussion Besides dramatic inhibitory effects on the release of GH from somatotrophs, somatostatin blocks the release of TSH, prolactin and ACTH from the pituitary. However, somatostatin has not been reported to affect gonadotrophin release (Reichlin, 1983; Brazean et aI., 1973). In our experiments we demonstrated that repeated ICV bolus injections of SRIF-14 or SRIF-28 changed directly the morphometric and immunocytochemical behaviour of LH gonadotrophs. The number of immunopositive LH cells per unit area

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was significantly decreased in both somatostatin-treated groups while the number of FSH-positive cells was similar to that in the controls. Stereological analyses revealed that the relative volume density of LH cells in both somatostatin-treated groups was significantly lower than in the controls. In this respect immunopositive FSH cells showed no difference in the treated rats. Volume densities of perykarya of FSH- and LH-positive cells were decreased in all treated females, being extremely different in LH-positive cells after SRIF-14 administration. The differences were significantly more pronounced in LH than in FSH-positive cells when compared to the controls. Our data are in accordance with those of Yu et al. (1994) who reported somatostatin inhibition of LH, but not of FSH release in male rats. These

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Fig. 2 a. Number of immunoreactive FSH and LH cells (No) per unit area (!!!m 2 ) in control (C) and female rats treated with SRIF-14 or SRIF-28. 2b. Cellular (Vc) volume (11m3) of FSH- and LH-immunopositive cells of control and somatostatin-treated females. 2e. Relative volume density (Vvc) of FSH- and LH-immunoreactive cells expressed in percentage (%) of total pituitary cells in mm 3. All values are means ± SEM; * P < 0.01; ** P < 0.005; *** P < 0.001

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authors also showed suppression of LHRH-induced LH release by somatostatin and no effect on basal release of either gonadotrophin and suggested that somatostatin inhibited LHRH activity by binding to somatostatin receptors on LH gonadotrophs. O'Carroll (1995) found mRNA of all five somatostatin receptor subtypes (rsstr 1-5) in somatotrophs, thyrotrophs, mammotrophs, corticotrophs and gonadotrophs of the anterior pituitary. The mRNA of rsstr 1 and rsstr 2 was present primarily in· the LH cells, whereas that of rsstr 3 predominated in FSH cells. Somatostatin regulates LH function through interaction with either rsstr 1 or rsstr 2 or through both of these receptors, whereas FSH interacts with rsstr 3 only. The affinity of somatostatin for the rsstr 3 receptor is significantly lower than for rsstr 1 and rsstr 2. In this respect we suggest that the concentration of somatostatins used in our experiments was high enough to affect the LH cells but too low to induce changes in the FSH gonadotrophs and a response to somatostatin respectively. This interretation is supported by the data of Samuels et al. (1992) who suggested that somatostatin can act in vivo to suppress the pituitary response to LHRH, since it suppresses the amplitude of LH pulses, but not that of FSH in normal humans. It has also been reported that gonadotrophin-releasing hormone-induced LH secretion was reduced significantly by somatostatin in normal menstruating women, whereas the concomitant FSH response was unaffected (Chiodera et al., 1986). PreleviC et al. (1990) observed that octreotide (Sandostatin®), a stable somatostatin analogue decreased the circulating concentrations of LH and ovarian steroids in women with polycystic ovarian syndrome (PCOS). On the other hand, Reubi et al. (1987) reported the presence of somatostatin receptors in the human pituitary tumor, gonadotrophinoma. Sy et al. (1992) showed reduction of gonadotrophin-secreting adenomas by treatment of patients with agonist somatostatin analogues. All these data demonstrate that somatostatins which may suppress the response of LH-gonadotrophs to LHRH have some clinical relevance. In conclusion, our results indicate that ICV-applied somatostatins exert significant inhibitory effects on the immunocytochemical and morphometric characteristics of LH, but not of FSH gonadotrophs. However, elucidation of the exact mechanism(s) underlying somatostatin action on gonadotrophic function in our experimental conditions requires further study.

Acknowledgments The authors wish to thank Dr Jelena Joksimovic of the Institute for Biological Research for her continuous interest in this work and useful suggestions. The kind help of Ms Ann Nikolic, Ph. D. of the Institute for Nuclear Energy Application, during the preparation of the manuscript is also appreciated. The authors are grateful to Dr A. F. Parlow, National Institute of Health, Bethesda, MD, USA, for the kind donation of the antisera. This work was supported by the Ministry for Science and Technology of Serbia, contract # 03E17.

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