Effect of kisspeptin on testes and accessory sex glands in prepubertal rats

Abstract

Hypothalamus-deri ved kisspeptins arc critical regulators of reproduction in nearly all mammalian species including the humans. These sma ll pcptides mediate their actions through the GIlRH loop system. How lcisspeptins regulate gonadal maturation in sexually immature male mammals remains elusive. To address this, two sets of experiments were done: firstly, kisspeptin was administered as subchronic (12 days) tw ice daily,i. p. do se~ at three different dosage regimens: IS pmol (10 pg). 1.5 nmol (I ng) and 1.5 )llnol (I )lg), to prepubertal male Sprague Dawley rats (PND 35), secondly, kisspeplin effects were further evaluated indirectl y by blocking GnRH action using acyline as an antagonist. Rats were assigned to four experimental groups namely: control saline treated, kisspeptin alone, saline with acyline pretreatment and kisspeptin with acyline pretreatment. Effects on spennalogenesis, secretion of testosterone and pituitary gonadotropins, the LH and FSH, DNA parameters and histomorpllology of testicular tissue and accessory sex glands, seminal vesicle and prostate, were studied. Major scientific approaches applied were; radioimmunoassaYl light and electron microscopy, DNA extraction, electrophoresis and morphomelricaI measurements. Spennalogenesis was studied histologicall y at stage VII of the spermatogenic cycl,e. Data were analyzed statistically. Resu.lts showed that at end of the treatments plasma FSH levels were not altered in any of the treatment groups. LH and testosterone concentrations were reduced in the I ng (p < 0.05) and I )lg kisspeplin groups (p < 0.01), whil e no significant change was observed at 10 pg dose. At I ng and I p.g kisspeptin doses, testicular parameters that decreased significantly were mainly: the number of type A spennatogonia (p < 0.05; P < 0.01), prcleptotene spennatocytes (p < 0.05), pachytene spemlatocytes (p < 0.01: p < 0.001), step 7 spennatids (p < 0.05;p < 0.00l), elongated spermatids and daily sperm production (p < 0.05; p < 0.00 I). while at 10 pg dose the decrease was non. significant. Sertoti cell efficiency and total support capacity of each Sertoli cell decreased significantly at all doses. Meiotic index decreased (p < 0.05) only at 1 J.Lg dose; while coefficient of mitosis increased at I ng and I Jlg (p < 0.0 I) doses both. Histomorphology showed scant round and elongated spermatids, intratubular vacuol izationst multinucleated giant cells and atrophied germinal epithelium. Ultrastmclure evidenced vacuo lated mitochondria in Serloli cells! XIII involuted acrosome, degenerated and vacuolated Leydig cells and thin basal laminae. DNA ladder assay showed fragmentatio n of DNA i.nto smaller fragmen ts of variable sizcs. On quantification, DNA damage that occurred to the testicular tissue was 20 ± 2.04,36 ± 1.85 and 60.18 ± 3.37, at 10 pg. I ng and I ~g doses respectively. [n tile second set ofexpcli ments plasma FSH (p < 0.00 1) and LH (p < 0.001) decreased in the saline (acyline pretreated) treated group, and as well as in the kisspeptin (acyline pretreated) treated group at the end of the experiment. Testosterone levels dec reased (p < 0.01) in saline (acyline pretreated) and kisspeptin (acyline pretreated) treated groups , (p < 0.001). Seminiferous tubular diameter decreased non-significantly while seminife rous tubular epithelial height decreased highly significantly (p < 0.001) in treated groups as compared to controL In saline (acyline pretreated) treatcd group, type A spermatogonia (p < 0,001), preleptotene spermatocytes (p < 0,05), pacbytene spermatocytes (p < 0,01). step 7 spermatids (p < 0,001), elongated spermatid head counl and daily speml production (p < 0.0 I) decreased as compared to control testes. In the kisspeptin (acyline pretreated) treatment group, only type A spermatogonia (p < 0.00 1), decreased significantly, while other parameters remai.ned unaffected, Histomorphoiogical observations demonstrated loss of genn cells, abnormal geml cell associations and gel'IYl l:t:il maturation an-est in saline (acyline pretreated) treated group, while seminiferous tubules of rats treated with kisspeptin (acyline pretreated.) revealed partial restoration of spermatogenesis, DNA damage to the testicular tissue was 36 ± 0.07 and 14 ± 1.1 7 with saline (aeyline pretreated) and kisspeptin (acyline preu'eated) treatment respectively, Seminal vesicle weights decreased significantly (p < 0,01) at I J.Lg kisspeptin dose, The epithelial beight of secretory acini of seminaJ vesicle decreased at 10 pg (p<O.05), 1 I1g and IJLg doses (p < 0.00l), Histological observations demonstrated dilated lumen and decrease in epithelial folds and height of epithelial cells. Ultrastructure showed disorgani zation of the organelles in volved in the secretory process such as ~tation qf the endoplasmic reticulum, disorganization of the GoJgi complex and decrease in the number of secretory granules in principal ceLIs of the seminal vesicle, DNA damage to the seminal vesicle was 19.54 ± 1.98, 38.06 ± 2.09 and 58. 18 ± 2.59 at to pg, I ng and I /lg doses respectively. [n the second set of experiments seminal vesicle weights remained unaffected, Epithelial height of secretory acini of seminal vesicle decreased (p < 0.05) in both the saline (acyline XI\' pretreated) and kisspeplin (acyli ne pretreated) treated groups as compared to the control. Histological and ultrastructural examination showed degeneration of the tissue in saline (acyl ine pretreated) trea ted group, while it nearly restored to nonnal in the kisspeptin (acyline pretreated) treated gTOUp. DNA damage was 20.87 ± 0.98 and 19.03 ± 1.7 1 with saline (acyli ne pretreated) and kisspcptin (acyline pretreated) treatment respect ively. Prostate weights decreased significantly (p < 0.05) at 1 p.g treatment dose of kisspepti.n. The epithelial height of sec.retory acini of prostate decreased at 10 pg (p < . . 0.05), 1 ng and lJ,tg doses (p < 0.001). Histology and ultrastructure demonstrated, decrease in epithelial cell height, epithelial folding and dilatation of the organelles with kisspeptin treatment. DNA damage to the prostatic tissue was 20.74 ± 2. 18, 43.60 ± 2.39 and 58. 18 ± 2.59 at 10 pg, I ng and 1 J.!,g doses respectivety. In the second set of experiments, prostate weights were unaltered. Epilhelial height of secretory acini of prostate gland decreased (p < 0.001) in saline (acyline pretreated) and (P < 0.05) in kisspeptin (acyline pretreated) treated groups. Histologically, the 'prostate tissue was degenerated and regressed with saline (acyline pretreated) treatment but showed a restoration of structure to nonnal with kisspeptin (acyliJle pretreated) treatment. DNA damage to the gland was 26.60 ± 1.71 and 14.02 ± 1.27 with saline (acyline pretreated) and kisspeplin (acyline pretreated) treatments respectively. The present findings indicate that subchronic kisspeptin administration may act as a suppressor of pubertal maturation during non-pubertal states and a partial recovery of the testi cular tissue and accessory sex glands occm with acyline pretreatment.