International Journal of Anatomical Sciences 2011, 2(1):11-17

Research Article

Long-Term Hyperglycemic effect on rat Epididymis and Sperm

Suresh S, Shanthi Santhosh Kumari S, Preethi U, Venkatalakshmi N, Karthik Ganesh M,Ganesh L, Prithiviraj E, Prakash S.

Department of Anatomy, Dr. Arcot Lakshmanasamy Mudaliar Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, INDIA.

Key Words: hyperglycemia, epididymis, sperm

Abstract: The epididymis, an important organ of male reproduction, imparts sperm maturation acquiring motility and fertilizing potential and removal of defective sperm and providing good microenvironment. In addition the epididymis plays a significant role in the transport, concentration, protection, and storage of spermatozoa. The objective of the present  study was to analyse the patho-physiological changes in epididymis, sperm morphology, count, maturation and transport under long-term hyperglycemic condition using Wistar Albino rats as diabetic (hyperglycemic) model. Twenty male Wistar albino rats (Rattus norvegicus) were used for this study (body weight 225– 250 g) The rats were randomly selected for the following groups i.e. group I – control (received 0.1M Citrate buffer) and group II – Diabetes (single injection of streptozotocin 60 mg/kg b.w. in 0.1M citrate buffer). At the end of 120 days animals were sacrificed by over dose of anesthesia (i.p), and followed by transcardial  perfusion  using 4  % paraformaldehyde  in  0.1  M  phosphate buffered saline. Epididymides were dissected out and after gross measurements were taken, processed  for paraffin technique. Sections  were stained using hematoxylin  (Harris’s) and eosin and observed under light microscope and histomorphometry and stereological analyses were done. Spermatozoa were collected from caudal portion of epididymis to estimate sperm viability and motility, morphology and morphometry, cytoplasmic droplets and epididymal transit time. The present data revealed major damage in structure of epididymis and altered sperm quality and quantity under the influence of long-term diabetes. The alteration in epididymis might have influenced the sperm damage and poor sperm parameters leading to infertility. With the age limit to acquire diabetes is coming down rapidly and thus increasing the risk of infertility in younger generation. Consequently, more number of basic and clinical researches should be focused on the prevention and cure of diabetes.

Infertility in males is a topping list of male related sexual problems nowadays. It affects as many as one in six couples, the possible causes comes under the following

Idiopathic, Varicocele, ageing, hormonal imbalance, Substance Abuse (alcohol, nicotine), Testicular factors, sedentary lifestyles, environmental factors, thyroid disease, hypertension, heart disease and diabetes  mellitus  (DM).  DM  may  affect male reproductive function at multiple levels including spermatogenesis as a result of its endocrine impairment (Baccetti et al., 2002; Ballester et al., 2004). Animal studies using rodent models of streptozotocin-induced diabetes   mellitus   have   demonstrated   a reduction in sperm count and quality (Ballester et al., 2004; Amaral et al., 2006; Scarano et al., 2006). Human diabetic study showed  reduction  in  all  semen parameters (semen volume, sperm count, motility and morphology) (Garcia-Diez et al., 1991).

The epididymis, an important organ of male reproduction, imparts sperm maturation acquiring motility and fertilizing potential  and  removal  of  defective  sperm and providing good microenvironment (Turner, 2007). In addition to sperm maturation,  the  epididymis  plays  a significant role in the transport, concentration, protection and storage of spermatozoa   and all these process are androgen dependent (Meistrich et al., 1975).

The  objective  of  the  present  study was to analyse the patho-physiological changes in epididymis and the sperm morphology, count, maturation and transport under long-term hyperglycemic condition using Wistar Albino rats as diabetic (hyperglycemic) model.

Materials and Methods

Animal Used

Twenty male Wistar albino rats (Rattus norvegicus) were used for this study. Male rats of body weight (b.w.) 225– 250 g were  selected.  They  were  housed individually in separate standard cages and maintained under standard laboratory conditions (temperature 24–28°C, relative humidity 60–70%, and 12-hour light–dark cycle) with free access to solid pellet diet and water ad libitum throughout the study. The study was approved by Institutional Ethical Committee. Quarantine procedures and  animal  maintenance  was  according  to the recommendations of Canadian Council Guide to the Care and Use of Experimental Animals (1993) and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines for laboratory animal facility (2003).  Experimentation  on  these  animals carried  out  after  subjecting  to  quarantine period of not less than 15 days each.

Experimental   Design   and   Induction   of  Diabetes

In this present study, animals were grouped as, group I – control (received 0.1M Citrate  buffer)  and  group  II  –  Diabetes (single injection of streptozotocin (Sigma- Aldrich,  USA)  60  mg/kg  b.w.  in  0.1M citrate buffer). At the 4th day after injection the animal blood glucose level were estimated.  Animals  which  showed  above 250   mg/dl   were   considered   as   diabetic (Suresh and Prakash, 2010).

Tissue Harvesting

At   the   end   of   120   days   after recording final body weights, animals were sacrificed by over dose of anesthesia (i.p), and followed by transcardial perfusion using 4 % paraformaldehyde in 0.1 M phosphate buffered  saline.  Epididymides  were dissected out and gross measurements were taken. The tissues were fixed for histological studies in freshly prepared Bouin’s fluid and processed for paraffin technique. Sections were  stained  using  hematoxylin  (Harris’s) and eosin and observed under light microscope (Nikon. Japan)

Sperm Analysis

Sperm count:

Sperm count was done according to the procedure described previously (Suresh et  al.,  2010).   Briefly  spermatozoa  were collect from caudal portion of epididymis, by mincing caudal epididymis with anatomical scissors in 5 ml of pre warmed (35º C) physiological saline, placed in a rocker for 10 min. and incubated at room temperature for 2 min. Supernatant fluid was diluted 1:100 with solution containing 5 g Sodium bicarbonate, 1 ml formalin (35 %) and 25 mg Eosin/ 100 ml H2O. Total sperm count  was  determined  using haemocytometer. Approximately 10μl of diluted sperm suspension was transferred to each counting chamber and was allowed to  stand for 5 min and then counted under a light microscope at 400 X magnification.

Sperm Viability and Motility:

About 20μl of sperm suspension was mixed with an equal volume of 0.05% eosin- Y and nigrosin. After 2 min incubation at room temperature, slides were viewed by bright-field microscope with 400 X magnification (Nikon, Japan). Dead sperms appeared pink and live sperms were not stained (Suresh et al., 2010).  Percentage of motile sperm was assessed using graded semi-quantitative scale of 0 to 5 and the spermatozoa were evaluated for the rate of forward movement and graded accordingly, i.e., 0 = No movement, 1= Sluggish or tail movement alone, 2 = Intermittent sluggish movement, 3 – 4 = Fair & Good movement and 5 = Maximum movement in forward direction.

Morphology and Morphometry:

The fixed sperm were smeared on a glass slide and stained with phosphate buffered saline solution of Giemsa (Merck, Germany) (Hafez, 1977). Morphological alterations  in  sperm  and  morphometrical data  acquired  by  measuring  length  of  the head and flagellum were evaluated. A total of one hundred spermatozoa were analyzed per animal using ocular micrometer scale fitted to a light microscope under 40X magnification.

Cytoplasmic Droplet Containing Sperm

The presence of cytoplasmic droplet containing sperm was determined using the method of Syntin & Robaire (2001). Spermatozoa were assessed for the presence or absence of cytoplasmic droplet, for which minimum of 100-spermatozoa/ animal were evaluated.

Estimation of Epididymal Transit Time

The epididymal transit time were calculated according to the methods of Scarano  et  al.,  (2006).  Briefly  the epididymal sperm count were calculated in individual  region  (Caput,  corpus  and caudal).  Then  transit  time  of  caput/corpus and caudal epididymis were calculated by dividing the number of sperm within each of these three regions by the daily sperm production.

Histomorphometry  and  stereological analyses

The conventional stereological principles and accepted morphometric procedures as outlined by Elias and Hyde, (1980) were used to obtain quantitative information, details of the procedure have been  described  previously  (Prakash  et  al., 2008).  Systematic   unbiased  random sampling (SURS) protocol was adopted. All the data’s are expressed in relative value.

Statistical analysis

The  significant  difference  between the mean value of control and experimental groups was  determined by student‘t’  test. P value <0.05 was considered as statistically significant (Zar, 1974). All the analysis was done by using Microsoft Excel 2003 and SPSS statistical package version 7.

Observations

Sperm analysis

Significant  reduction  in  percentage of  viable  and  increase  of  the  dead  sperm were observed in the long term hyperglycemic rats when compared with normoglycemic rats.  There was  significant decline in motility in hyperglycemic when compared with normoglycemic rats.

The  sperm  concentrations  and motility  were  markedly  decreased  in  the long term hyperglycemic rats. No significant changes were observed in the morphometry study in all the experimental groups. Morphological analysis shows wide degree of  abnormality  in  long  term  diabetic  rat sperm such as defects in the head (microcephalic,  bicephalous,  amorphous, and acephalic), neck and tail when compared to the group I.

Cytoplasmic droplet containing sperm

Cytoplasmic droplet study showed significant increase of the sperm containing cytoplasmic droplets in long term hyperglycemic rats (45 ± 1.12) when compared to normoglycemic rats (3 ± 0.39). These cytoplasmic droplets were located at various levels in the sperm; tail remnant was more in number. Observations are summarized in table 2.

Epididymal transit time

Significant reduction in the epididymal transit time was observed in the long term diabetic rat when compared with normal rats. Data’s were shown in table 1.

Table 1 Sperm analysis data and epididymal transit  time  of  control  and  long term diabetic animals. Each value indicates the mean ± SD of (n – 6) animals. a – control, *** – p<0.001.

PARAMETER CONTROL DIABETES
Sperm viability (%)
Live 98 ± 1.23 60 ± 2.12 a***
Dead 2 ± 0.89 40 ± 1.65  a***
Motility (%) 98 ± 2.15 50 ± 1.86 a***
Count (106) 371 ± 12.02 128 ± 3.25 a***
Morphology (%)
Normal 96 ± 2.12 67 ± 3.20 a***
Abnormal 4 ± 0.35 33 ± 1.32 a***
Epididymal sperm count (X106/organ)
Caput/ Corpus 123.67 ± 4.68 45.23 ± 2.17 a***
Caudal 247.33 ± 2.63 82.77 ± 1.86 a***
Epididymal sperm  transit time (days)
Caput/ Corpus 6.110 ± 0.25 2.47 ± 0.86 a***
Caudal 12.22 ± 1.01 4.52 ± 1.06 a***

Epididymal Morphological Changes

There were morphological alteration such as decrease in the length, breadth, height, weight and volume in all the three regions of epididymis i.e. caput, corpus and cauda of long term hyperglycemic rats when compared to the normoglycemic  rats. Data’s were shown in table 2)

Table  2  Morphology of  epididymis  in  control and long term diabetic animals. Each value indicates the mean ± SD of (n –6) animals. a – control, * – p<0.05, ** -p<0.01 and *** – p<0.001

 

PARAMETER Control Diabetes
Length (cm) 5.33 ± 0.24 4.1 ± 0.15 a **
Breadth (cm)
Caput 0.63 ± 0.12 0.25 ± 0.11 a**
Corpus 0.3 ± 0.10 0.13 ± 0.09 a*
Caudal 0.85 ± 0.08 0.39 ± 0.11 a**
Width (cm)
Caput 0.53 ± 0.12 0.15 ± 0.09 a***
Corpus 0.15 ± 0.03 0.1 ± 1.59 a*
Caudal 0.54 ± 0.06 0.24 ± 0.08 a**
Volume (ml) 0.48 ± 0.11 0.15 ± 0.09 a**
Weight (gm) 0.643 ± 0.06 0.31 ± 0.03 a***

Histological observation

Epididymal histology shows the thickening of basement membrane, vacuolation   of   the   epithelial   cells   and absence of mature spermatozoa and presence of  immature  germ  cell  in  the  lumen  of tubules in the long term diabetic animals when compared to the control (Fig. 1).

Histomorphometric Changes

There was significant change in volume of connective tissue, tubule and epithelia. The number of tubule was found to be increased and the diameter found to be decreased in diabetic group (Figure 2 to 7).

Discussion

The   present   data’s    revealed    that major changes in structure and functions of epididymis   plays   a   significant   role   in altering sperm quality and quantity under the influence  of  long-term  diabetes.  This includes reduction in gross weight and volume of the epididymis concomitant with decrease in the volume of spermatozoa in the tubular lumen may be partly accounted for the reduction in epididymal weight and volume in the diabetic rats. This may be due to the impaired spermatogenesis in diabetic condition (result not shown).

Fig. 1 Morphology and histology of the epididymis in control and long term diabetic rats. In diabetic animal (arrow) showing vacuole formation in the epithelium Arrow head indicates the immature germ cells in the lumen and the star indicates the occluded lumen.

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Fig.  2  Diameter  of  the  epididymal  tubules  of control and long term diabetes animals.

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Fig. 3 Height of the epididymal epithelium of control and long term diabetes animals. Each bar indicates mean ± SEM of (n-6) animals. a – control, * – p< 0.05, ** -image004

 

 

 

 

 

 

 

Fig. 4 Volume of connective tissues of control and long term diabetes animals. Each bar indicates mean ± SEM of (n-6) animals. a

 

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Fig. 5 Volume of epididymal epithelium of control and long term diabetes animals. Each bar indicates mean ± SEM of (n-6) animals. a – control, *** – p< 0.001.

 

 

Fig. 6: Volume of epididymal tubules of control and long term diabetes animals. Each  bar indicates mean ± SEM of (n-6)animals. a – control, ** – p<0.01 and *** – p< 0.001. shows that epididymal dysfunction and disturbed homeostasis. The pathology could have been enhanced by the impaired spermatogenesis and immature spermiation which produce immature or sperm with cytoplasmic  droplets  (Suersh  and  Prakash,2010).

The   changes   in   epididymis   had severely  affected  sperm  parameters  when compared to control. The sperm count and motility were decreased with increased number of abnormal sperm in the hyperglycemic epididymis. The sperm with  Fig 7 Number of the epididymal tubules of control and long term diabetes animals. Each bar indicates mean ± SEM of (n-6) animals. a – control, * – p<0.05 and *** – p< 0.001. cytoplasmic droplet was also more in hyperglycemic rat indicating patho- physiological state of the testis and epididymis. The presence of sperm with cytoplasmic droplet can increase free radical production and oxidative stress in the epididymis, a condition seen in our previous  study on aged rat epididymis (Suresh et al.,2010).

 The histological observation of diabetic epididymis showed vacuolation, epithelial fibrosis and lumen filled with immature and degenerative sperm cells, demonstrating the deleterious effect of long-

In general epididymis depends on androgens to maintain their structure and function (Amman et al., 1993). The morphological alteration seen in diabetic epididymis may be predominantly due to the androgen deficiency.   The serum testoste- rone and luteinizing hormone were reduced in diabetic condition as observed in our previous study (Suresh and Prakash, 2010). Usually in normal rats deprived of testosterone, there will be increase in the rate of epididymal sperm transit time (Meistrich et al., 1975). However, in this study the diabetic rats deprived of testosterone showed significant  decrease  in  epididymal  transit time for sperm. There was increased amount of  immature  and  abnormal  sperm  in  long term diabetic rats when compared with control  rats.  Present  observations  clearly  term hyperglycemia. Histomorphometeric observations  showed  more number of tubules per unit area. This might be due to the shrinkage of tubular diameter. Similarly this shrinkage or concentration of more tubules might have caused increase in epithelial height and volume. There was increased amount of connective tissue proportion.

 These structural changes suggested that the epididymal physiology could have severely altered and consequently, resulted in sperm damage which may lead to infertility. With the age limit to acquire this disease is coming down rapidly and thus increasing the risk of infertility in younger generation. Given the finite nature of the human existence,  an individual  man’s  most precious and lasting gift to the world is the legacy created by his  offspring.  The most unfortunate thing is that  India will be the diabetes capital of the world in 2030 according to  a World Health Organization (WHO) report.   Hence, more basic and clinical  research  is  required  for  the prevention and cure of diabetes in India.

References

Ali  ST,  Shaikh  RN,  Siddiqi  NA  (1993)  Semen analysis in insulin-dependent/non-insulin- dependent diabetic men with/without neuropathy. Arch Androl, 30: 47–54.

Amaral   S,   Moreno   AJ,   Santos   MS,   Seica   R, Ramalho-Santas J (2006) Effects of hyperglycemia on sperm and testicular cells of Goto-Kakizaki and streptozotocin-treated rat models for diabetes. Theriogenology, 66: 2056–2067.

Amman   RP,   Hammerstedt   RH,   Veeramachaneni DNR      (1993)    The    epididymis   and    sperm maturation: a perspective. Reprod Fert Dev, 5:361–381.

Baccetti B, Marca Al, Piomboni P, Capitani S, Bruni E, Petraglia F, De Leo V (2002) Insulin- dependent diabetes in men is associated with hypothalamo-pituitary derangement and with impairment in semen quality. Hum Reprod, 17:2673–2677.

Ballester J, Muñoz MC, Domínguez J, Rigau T, Guinovart JJ, Rodríguez-Gil JE (2004) Insulin- dependent diabetes affects testicular function by FSH- and LH-linked mechanisms. J Androl, 25:706–719.

Elias H, Hyde H (1980) An elementary introduction to stereology. Am J Anat, 159: 411–446.

Cameron DF, Rountree J, Schultz RE, Repetta DF, Murray   T   (1990)   Sustained   hyperglycemia results in testicular dysfunction and reduced fertility potential in BBWOR diabetic rats. Am J Physiol, 259: E881–E889.

Committee  for  the  Purpose  of  Control  and Supervision on Experiments on Animals CPCSEA    guidelines for laboratory animal facility (2003)   Committee for the Purpose of Control and Supervision on Experiments on Animals. Ind J  Pharmacol, 35: 257–274.

Olfert ED, Cross BM, McWilliam AA (1993) Guide to the care and use of experimental animals. Volume 1. 2nd ed. Canadian Council for Animal Care.

Frenkel GP, Homonnai ZT, Drasnin N, Sofer A, Kaplan  R,  Kraicer  PF  (1978)  Fertility  of  the

streptozotocin-diabetic male rat. Andrologia, 10:127–136.

García-Díez LC, Corrales Hernandez JJ, Hernandez- Diaz J, Pedraz MJ, Miralles JM (1991) Semen characteristics and diabetes mellitus: significance of insulin in male infertility. Arch Androl, 26:119–128.

Hafez ESS (1977) Techniques of human andrology.

Published       by       Elsevier/North-       Holland    Biomedical press. 471. Amsterdam, Netherland.

Handelsman DJ, Conway AJ, Boylan LM, Yue DK, Turtle   JR   (1985)   Testicular   function   and glycemic control in diabetic men. A controlled study. Andrologia, 17: 488–496.

Meistrich ME, Hughes TH, Bruce WR (1975) Alteration of epididymal sperm transport and maturation  in  mice  by  oestrogen  and testosterone. Nature, 258: 145-147.

Mori H, Christensen AK (1980) Morphometric analysis of Leydig cells in the normal rat testis. J Cell Biology, 84: 340-354.

Phil-Ok Koh (2007) Streptozotocin-Induced Diabetes Increases Apoptotic Cell Death in Rat Epididymis. Lab Anim Res, 23: 381-384.

Prakash  S,  Prithiviraj  E,  Suresh  S  (2008) Development of seminiferous tubules in Prenatal, Postnatal and adult Testis of Bonnet Monkey (Macaca radiata). Anat Hist Emb, 37: 19 – 23.

Syntin P , Robaire B (2001) Sperm structural and motility changes during aging in the brown Norway rat. J Androl, 22 : 235–244.

Scarano WR, Messias AG, Oliva SU, Klinefelter GR, Kempinas WG (2006) Sexual behaviour, sperm quantity and quality after short-term streptozotocin-induced  hyperglycaemia  in  rats. Int J Androl, 29: 482–488.

Sexton WJ, Jarow JP (1997) Effect of diabetes mellitus upon male reproductive function. Urology, 49: 508–513.

Suresh S, Prithiviraj E, Prakash S (2010) Effect of Mucuna pruriens on oxidative stress mediated damage in aged rat sperm. Int J Androl, 33: 22-32.

Turner TT (2008) De Graaf’s Thread: The Human Epididymis. J Androl, 29: 237-250.

Vignon F, Le Faou A, Montagnon D, Pradignac A, Cranz C, Winiszewsky P, Pinget M (1991) Comparative study of semen in diabetic and healthy men. Diabete Metab, 17: 350–354.

Zar  JH  (1974)  Biostatistical analysis.  Engle  wood cliffs NJ; Prentice Hall Inc.