Egypt. J. Histol. Vol. 31, No.2, Dec.

2008: 354 - 361

(ISSN: 1110 - 0559)

Original Article Effect of Short Photoperiod on the Leydig Cells of Rat's Testis Ultrastructural and Immunohistochemical Study
Nagwa A. Ahmed
Histology Department, Faculty of Medicine, Cairo University

ABSTRACT
Purpose: This study aimed to investigate by using electron microscope and immunohistochemistry, effects of short duration of light exposure on the Leydig cells of rat's testis. It tries to relate between seasonally changed environmental light factor and an important part of male reproductive system. Methods: Sixteen adult male albino rats were divided according to light exposure into control group (six rats) exposed to light: dark 12:12 h and group II (ten rats) exposed to light: dark 8:16h, daily for eight weeks. Light was provided by 20 watt fluorescent bulb. Testes Specimens were prepared for electron microscope and paraffin sections were stained for Fas and VEGF immunohistochemistry. Optical density of VEGF immunoreaction in Leydig cells was assessed morphometrically. Results: In group II, the ultrastructural findings showed less mitochondria, smooth endoplasmic cisternae and secretory vesicles. Cytoplasmic vacuoles and irregularity of cell membrane of some Leydig cells were detected. Irregular nuclear membrane and condensation of nuclear chromatin were seen. Immunoreactivity for Fas was positive in some Leydig cells of group II. VEGF immunoreaction was decreased in many Leydig cells and this was proved by a significant decrease in mean optical density of VEGF immunoreaction. Conclusion: Short photoperiod exposure (which reflects short days of the year) may result in hypofunction of the Leydig cells in rat's testis with possible consequent decreased testosterone secretion and reproductive regression. So, seasonal fluctuations in Leydig cells morphology and function may serve to synchronize reproduction with favorable environmental conditions. Key Words: Short photoperiod, leydig cells, fas, VEGF. Corresponding Author: Nagwa Abdel Wahab Tel.:0106637892 E-mail: nagwahistology2002@yahoo.com

INTRODUCTION
The photoperiod is the cycle of light and darkness to which an animal or plant is exposed each 24-hours period. Natural photoperiods follow the natural cycle of day and night where daylight is the only source of illumination. This natural cycle is also changing with the season, providing short days (short photoperiods) in the winter and long days (long photoperiods) in the summer1. Photoperiod effects are aspects of chronobiology that is the science of biological cycles or circadian rhythm. Most our physiology, behavior and biochemistry is rhythmic, showing day-night differences2. Although environmental variables, as temperature and humidity, affect the body physiology and fluctuate on a seasonal basis, but changes in the ambient photoperiod provide the most reliable effects3. Exposure to light above or below a critical value may have unintended effects on the endocrine and metabolic states which might affect most organs like liver and kidney 4 as well as cartilage and bone metabolism5. Also, it causes large shift in body mass, thermoregulation, immune function and general activity6. Male and female genital systems are the most affected in photoperiod variations or day length. Seasonal changes from summer to winter is an important physiology trait that modify reproductive timing in many temperate zone mammals including sheep, horses and rodents7. The present study aimed to investigate by electron microscope and immunohistochemistry for Fas and VEGF (vascular endothelial growth factor), the effects of short duration of light exposure on the interstitial cells of Leydig in rat's testis. It tries to relate between seasonally

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changed environmental element (light) with an important part of reproductive system (Leydig cells). MATERIALS AND METHODS In this study, sixteen adult male albino rats were used; their weight was ranging from 200 to 215grams. The rats were obtained from Animal House of Kasr El Aini, where they were kept under good hygienic conditions. They were housed in fan ventilated wide polypropylene cages with stainless steel tops and wood shavings for bedding. Food and water were available ad libitum. Temperature was maintained at 232c. Rats were divided according to light exposure into two groups; each group was kept for eight weeks in a separate cage: I) Control group: Included six rats, exposed daily to light from 7.00 am to 7.00 pm i.e. light: dark 12:12 h. II) Short photoperiod exposed group (Group II): Included ten rats exposed daily to light from 7.00 am to 3.00 pm i.e. light: dark 8:16h. Light was provided by 20 watt fluorescent bulb placed 5cm above the floor of cage (illuminance of 100 -250 Lux)8. The animals were sacrificed by decapitation; testes were carefully dissected and exposed to the following techniques: 1- Transmission electron microscopic examination: Specimens of right and left testes, 1 mm thick were fixed in 2.5% gluteraldehyde in 0.2 M phosphate buffer (pH 7.4) at 4C, postfixed in 1% osmium tetroxide. After dehydration in acetone, the specimens were embedded in Epon and cut using ultramicrotome. Semithin sections were stained with 1% toluidine blue for light microscopic examination and ultrathin sections were stained with uranyl acetate and lead citrate9 to be examined and photographed by Jeol 1010 Jam transmission electron microscope. 2- Immunohistochemical examination: Parts of right and left testes were fixed in Bouin's solution then processed; paraffin blocks were prepared and cut in serial sections of 5um thickness. Sections of right and left testes were stained by: A) Fas immunohistochemical staining: The Fas receptor (APO -1, CD95) is a type I membrane protein (45KDa) that can mediate apoptotic death in target cells, so it is used to detect apoptotic cells10. The primary antibody for Fas is mouse polyclonal antibody (Neo markers, Lab Vision Corporation); it was applied for 60 minutes on testis sections. Antigen retrieval was performed by placing slides in citrate buffer (pH 6.0). The primary antibody binding to tissue sections

was visualized with DAB. The reaction was observed as dark brown color in the cytoplasm of the Leydig cells compared with the positive control (prostate). Counterstaining was done using Mayer haematoxylin. Negative control was done without using the primary antibody11. B) VEGF immunohistochemical staining: VEGF (vascular endothelial growth factor) is a homodimeric glycopeptide that is a potent mitogenic growth factor for endothelial cells. The primary antibody to VEGF is a rabbit polyclonal antibody (Neo markers, Lab Vision Corporation) incubated at room temperature for 18 h with a dilution 1:600. VEGF antibody was detected using diaminobenzidine (DAB) substrate. The reaction was observed as grades of brown color in the cytoplasm of Leydig cells compared with positive control (The haemangiosarcoma). Counterstaining was done using Mayer haematoxylin. Negative control was done without using the primary antibody12. Scoring of the VEGF results was according to the following parameter: a. Negative > No reaction (compared with - ve control). b. Moderate > intermediate reaction. c. Strong > > intense reaction. (b & c compared with + ve control). 3- Morphometric study: Using Leica Qwin 500 LTD image analysis, the mean optical density of VEGF immunoreaction in Leydig cells in 10 high power (x400) fields using binary mode were assessed. Statistical analysis of the obtained data was done using Student's-t test. P (Probability) values <0.05 were considered significant.

RESULTS I) Electron microscopic results: Leydig cells belonging to control group (Group I) showed large rounded indented euchromatic nuclei, many mitochondria, well developed smooth endoplasmic cisternae and many secretory vesicles (Fig. 1). Following exposure to short photoperiod (Group II), the ultrastructure of rat Leydig cells showed rarified cytoplasm with less mitochondria, less secretory vesicles and few cytoplasmic vacuoles in some Leydig cells compared with control group (Fig. 2). Other Leydig cells showed irregular cell membranes. In addition, the nuclei appeared atypical in shape with

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Effect of Short Photopcriod on the Lcydig Cells of Rat's Tcstis Ultrastructural and Immunohistochcuiical Stud) peripheral condensation o f chromatin. The cytoplasm recruited less s m o o t h e n d o p l a s m i c cisternae in comparison t o control g r o u p (Fig. 3). Other fields revealed Leydig cells with the nuclei h a v i n g irregular nuclear m e m b r a n e , peripheral chromatin c o n d e n s a t i o n will) islands o f chromatin b e s i d e few o r g a n e l l e s in the c y t o p l a s m (Fig. 4). II) I m m u n o h i s t o c h e m i c a l results: A)Fas immunoreaction: I m m u n o h i s t o c h e m i c a l staining of r a t ' s testes sections in the control g r o u p revealed negative Fas cytoplasmic i m m u n o r e a c t i o n in t h e cells of Leydig (cells with rounded pale nuclei in close relation to blood vessels) (Fig. 5). Positive Fas i m m u n o s l a i n i n g was observed in the c y t o p l a s m of s o m e L e y d i g cells in short photopcriod e x p o s e d g r o u p ( G r o u p II) (Fig. 6). B)VEGF immunoreaction: I m m u n o h i s t o c h e m i c a l staining for V E G F (vascular endothelial g r o w t h factor) in the control lestis sections was positive in the cytoplasm of a g r o u p of L e y d i g cells (Fig. 7), m a n y cells expressed moderate reaction (Fig. 8). In other fields, strong cytoplasmic reaction was detected in occasional Leydig cells (Fig. 9). In rats e x p o s e d to short photopcriod. V E G F i m m u n o s t a i n i n g was negative in the c y t o p l a s m of m a n y Leydig cells (Figs. 10.11). In other fields, s o m e cells s h o w e d moderate cytoplasmic i m m u n o r e a c t i o n for V E G F (Fig. 12). T h e r e w e r e no differences in all p r e v i o u s findings appeared in the Leydig cells of both right and left rat's testes in control and experimental g r o u p s . M o r p h o m e trie results: M e a n optical density of V E G F i m m u n o r e a c t i o n was significantly decreased in g r o u p II (short photopcriod e x p o s e d rats) in c o m p a r i s o n to the control g r o u p (Table. 1) (Fig. 13). Table I: Mean optical density of VEGF immunoexprcssion SD in the Leydig cells in control and experimental groups. Groups
Group I (Contra))

Fig. J.1 Electron photomicrograph of a section in the lestis of a control rat showing a Lcydig cell with a large rounded dichromatic indented nucleus (N). many mitochondria (M), many smooth endoplasmic cisternae (f) and many secretory granules (S). Original magnification X 15000.

Mean optical density of VEGF imimuioreaction SD 0.78 0.02 Fig. 2: Electron photomicrograph ol a section in the lestis ol .i ral exposed to short photopcriod (Group II) showing ;i I cydig cell with a rounded nucleus |N). a rarilied cytoplasm dial shows few mitochondria (M) and secretory granules (Si. Noie a cytoplasmic vacuolc (V) i Iriginal magnification \ 12000

Group II (short photopcriod exposed rats)

0.61 i 0.01

Significant P<o.o5

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Fig. 3: Electron photomicrograph of a section in the rat's icslis of group II showing a Leydig cell with an atypical nucleus (N) and peripheral chromatin condensation. The cell membcrane is irregular: the cytoplasm appeared with few mitochondria (M). smooth cndoplasmic cisternae {\) and secretory granules (S). Original magnification X 15000.

Fig. 6: A photomicrograph of a section in the (estis of a rat exposed to short pholoperiod (Group II) showing part of seminiferous tubule (T) with positive reaction in the cytoplasm of some Leydig cells ( ) present in the inlersiiliiini. Fas immuLiostaining, X1000.

Pig, 4: Electron photomicrograph of a section in the rat's testts of a group II showing a nucleus of Leydig cell (N) with irregular nuclear membrane Fig. 7: A photomicrograph of a section in the teslis of a control ral showing and peripheral chromatin condensation with islands of condensed parts of three seminiferous tubules (T) with a positive reaction in the chromatin. few mitochondria (M) appealed in rarilied cytoplasm. cytoplasm of a group of Leydig cells (|) in the interstitium. Original magnification X 12600. VLGI" imniunoslaining X 400.

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Effect of Short Photopi-riod on the Leydig Cells of Rat's Testis Ultrastructurnl and liniminohistueheniical Study

Fig. 8: Higher magnification of the previous figure showing many Leydig cells in the interstitium.with moderate positive cytoplasmic reaction (f), VEGF immunostaining XIO00.

Fig. II: A photomicrograph of a section in the rat's testis of group 11 showing parts of two seminiferous tubules (T) with a negative reaction in the cytoplasm of two Leydig cells groups (f) around blood vessels (B). VEGF immunostaining X 1000.

Fig. 9: A photomicrograph ol a section in the testis ol a control rat showing a Leydig cell around a blood vessel (B) with a strong positive (A) and others with moderate cytoplasmic reaction (]). VEGF immunostaining X 1000.

Fig. 12: A photomicrograph of a section in the rat's testis of group II showing parts of two seminiferous tubules (T) with a moderate reaction in the cytoplasm of Leydig cells (f) beside blood vessel (B). Note negative reaction in some cells (D), VEGF immunostaining X 1000.

Fig. 10: A photomicrograph of a section in the testis of a rat exposed to short photoperiod (Group II) showing parts of seminiferous tubules (T) with a negative reaction in the cytoplasm of interstitial Leydig cells (J). Note blood vessels (B). VF-GF immunostaining X 400.

Fig. 13: Mean optical density of VEGF immunoexprcssion Leydig cells in control and experimental groups.

SI) in the

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DISCUSSION
The findings of this study demonstrated that short photoperiod induced damage ofthe interstitial Leydig cells of testis that might affect reproduction in rats as detected by electron microscopic and immunohistochemical studies. In this study, short photoperiod exposed rats showed few mitochondria, secretory vesicles and smooth endoplasmic cisternae. These ultrastructural findings could suggest hypofunction of the Leydig cells with consequent possible reduction of testosterone secretion. These mentioned results were consistent with a previously published study that recruited inhibited reproduction in many species during short days of winter13. Limiting reproductive activities to a specific season of the year prevents the production of offspring when ambient conditions are generally not favorable for the survival12. Other study demonstrated that testicular functions increase after exposure to long photoperiod and decreased after short photoperiod9. Also, it was stated that although short days enhance immune function by increasing leukocyte number and behavioral responses to infection, it induces gonadal regression and inhibits testosterone secretion14. The molecular mechanisms regulating seasonal photoperiod-induced testicular regression (atrophy) could be explained by the photoperiod pathway15. It transducers the photic information into a neural signal beginning from specialized photoreceptors in the eye to the suprachiasmatic nucleus of the hypothalamus and eventually passes to the pineal gland16. Absence of light results in pinealocytes activity and stimulates melatonin synthesis, by increasing the activity of biosynthetic enzymes that convert the serotonin into melatonin. For this reason, melatonin is secreted primarily and entirely in darkness and provides a neuro-endocrine representation of photoperiod17. The mechanisms by which melatonin adjusts seasonal reproductive activity in human is indirectly via the hypothalamo-pituitary axis by decreasing gonadotropin secretion. Also, melatonin acts directly at the gonads by reducing testosterone secretion from Leydig cells via two ways: decreasing cytosolic Ca^ release from intracellular stores of Leydig cells and through local inhibition of adenylcyclase (cAMP) activity18. Similar findings were reported in hamsters19. Another possible mechanisms by which melatonin may enhances short days - induced testicular regression is through adrenal hormones (especially of adrenocortical origin) via hypothalamo pituitary - adrenal axis, so adrenal hormones have been implicated in regressive behavior20. Increased regression displayed by melatonin- treated animals, however, can be blocked by adrenalectomy21. Cytoplasmic vacuoles detected in the cytoplasm of Leydig cells exposed to short photoperiod in this study,

can be explained by reduced activity of ATPase results in failure of sodium pump mechanism that responsible for water and electrolytes control. So, cells accumulate water that caused vacuolar degeneration22. Condensation of chromatin in the nuclei and irregular nuclear membranes appeared in the ultrastructural sections of rat's Leydig cells of group II, indicating apoptotic changes. It was proved that apoptosis is a well characterized form of cell death that is initiated by environmental cues like the darkness and involves the activation of specific genes that execute a linear cascade of cell death23. Short photoperiod induced testicular apoptosis may be due to deprivation of gonadotropins and reduction of testosterone2425. Apoptotic findings obtained by electron microscope were consistent with positive immunoreaction for Fas in the cytoplasm of rat's Leydig cells of group II. The possible involvement of the Fas system in the mediation of photoperiod-induced seasonal regression of Leydig cells was assessed using immunohistochemistry to localize Fas protein expression11. In the present study, VEGF immunoreactivity was detected in the cytoplasm of Leydig cells of control rats, indicating that this factor normally accumulates in these cells of the rat's testes. This was consistent with VEGF expression observed in the Leydig cells of humans26. It was proved that Leydig cells can synthesize and produce VEGF under hypothalamo-pituitary control. VEGF has a paracrine mitogenic function that causes continuous remodeling of the testicular microvasculature through formation of new blood vessels from existing capillary beds (angiogenesis) and stimulation of microvascular permeability27. Transfer to short photoperiod in group II resulted in reduced VEGF immunoreactivity in the cytoplasm of Leydig cells, confirmed by a significant decrease in its mean optical density. This was consistent with the study detected that reduced VEGF secretion in short days (under the effect of increased melatonin) may lead to vascular affection of testes with reduced oxygen and nutrient supply of testicular cells resulting in an increase of apoptotic cell death12. It could be concluded that exposure to short photoperiod (reflect short days of the year) resulted in hypofunction of the interstitial cells of Leydig in rat's testes. This was proved by reduced organelles, apoptotic nuclear changes evidenced by + ve Fas immunoreactivity and affected vasculature detected by decreased VEGF immunoreaction. So, seasonal fluctuations in Leydig cells morphology and functions may serve to synchronize reproduction with favorable environmental conditions. Further researches in human could be recommended to point the time of the year with best environmental

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conditions for the highest Leydig cells activity particularly in cases of in vitro fertilization, when best sperm activity is required.

13.

Jasnow AM, Huhman KL, Bartncss T.I and Demas GE. (2000): Short-day increases in aggression arc inversely related to circulating testosterone concentrations in male Siberian hamsters (Phodopus sungorus). Horm. Behav. Sep; 38 (2): 102-110. Prendergast BJ, Baillie SR and Dhabhar FS. (2008): Gonadal hormone-dependent and -independent regulation of immune function by photoperiod in Siberian hamsters. Am.J.Physiol. Regul.Integr. Comp. Physiol. Feb; 294 (2): R384-92.

14.

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