Status of Spermatogenesis and Sperm Parameters in Langur Monkeys Following Long-term Vas Occlusion With Styrene Maleic Anhydride

Authors


Reproductive Physiology Section, Department of Zoology, University of Rajasthan, Jaipur 302 004, India (e-mail: lohiyank@hotmail.com, lohiyank@rediffmail.com).

Abstract

ABSTRACT: Vas occlusion by styrene maleic anhydride (SMA), trade name RISUG (one of the promising male contraceptive procedures currently in phase III clinical trials), at 60 mg/vas deferens dissolved in 120 μL dimethyl sulphoxide (DMSO) at up to a 540-day study period caused severe oligospermia in the first 2 to 3 ejaculations and uniform azoospermia in the subsequent ejaculations without toxicity in langur monkeys. The ejaculated spermatozoa were necroasthenoteratozoospermic, suggesting instant sterility. Routine hematology and clinical chemistry parameters and the serum testosterone and sperm antibody titers remained unchanged from their pretreatment values until 540 days vas occlusion. Histology of testes revealed continued spermatogenesis throughout the study period. The stages of spermatogenesis appeared normal until 300 days of vas occlusion. At 360 days of vas occlusion, germ cells appeared in the lumen. Degeneration of seminiferous epithelium was evident in some of the tubules. Following 420 days of vas occlusion, the central portion of the testis showed regressed seminiferous tubules depicting various shapes and devoid of germ cells, which continued until 540 days of vas occlusion. Ultrastructure of the testes after 540 days of vas occlusion revealed vacuolization in the cytoplasm of Sertoli cells and degenerative features in the membranes of the spermatocytes and spermatids in the affected seminiferous tubules. The sub-cellular features of the normal tubules were similar to those of controls. The results suggest focal degeneration of seminiferous epithelium in the central portion of the testis following long-term vas occlusion with SMA.

Styrene maleic anhydride (SMA), an intravasal injectable male contraceptive currently undergoing phase III clinical trials, is a copolymer of styrene and maleic anhydride. It offers fertility control through multiple mechanisms in that it blocks the vas deferens, prevents the forward flow of sperm, and provides a pH-lowering effect and charge disturbances to the membrane of the spermatozoa, resulting in acrosome damage, which thus becomes deleterious to spermatozoa that may pass through the vas lumen (Guha, 1995, 1996). It has also been reported that SMA has no teratogenicity and is toxicologically safe in animal models (Sethi et al, 1990a,b,c). Clinical trials at phase I and phase II levels proved the safety and contraceptive efficacy in humans (Guha et al, 1993, 1997).

However, in order to make this procedure widely acceptable as a better alternative for vasectomy, its reversibility and related issues need to be addressed. Preliminary investigations for short-term reversal by a noninvasive approach have been successfully demonstrated in langur monkeys (Lohiya et al, 1998b; Mishra, 1999). The present investigation addresses the issues related to the long-term sequel of vas occlusion with SMA on the status of spermatogenesis, sperm antibodies, and serum testosterone in langur monkeys, an animal model closer to human in anatomy and reproductive exocrine and endocrine profiles (David and Ramaswami, 1971; Lohiya et al, 1988; 1998c).

Materials and Methods

Thirteen adult male langurs Presbytis entellus entellus were procured from places in and around Jaipur. The animals were maintained in the Department Primate Facility, under veterinary supervision, as per the Guidelines for Care and Use of Animals for Scientific Research, Indian National Science Academy (2000). Prior to the onset of the experiment, at least 3 pretreatment semen and blood samples that served as individual reference controls were collected. Both blood and semen samples were collected between 9 and 10 am throughout the course of the investigation.

Ten animals were anesthetized with sodium thiopentone at 20 mg/kg body weight intravenously. A small incision was made close to the external inguinal segment and the vas deferens was exposed from the spermatic cord. Sixty milligrams SMA dissolved in 120 μL of solvent vehicle dimethyl sulphoxide (DMSO) was injected into the lumen of the vas with the flow directed toward the ampulla of the vas deferens. Compression was maintained with the fingers, just distal to the injection site, to avoid retrograde flow. After injection, the spermatic cord sheath was closed with catgut suture and the skin with nylon. This procedure was performed bilaterally. Postoperative care was provided with antibiotics and anti-inflammatory drugs, and all the animals had uneventful postoperative recovery (Lohiya et al, 1998b; Mishra, 1999). Three animals served as sham-operated controls. The experiments were conducted in accordance with accepted humane practices.

The body weight; semen analysis (World Health Organization, 1999); status of serum testosterone (DPC, Markham, Ontario, Canada) and serum sperm antibodies (Bioserv AG, Rostock, Germany); and toxicological evaluation through routine hematology and clinical biochemistry (Reagent kits, Transasia Biomedicals Ltd, Mumbai, India) were assessed at monthly intervals up to the end of the 540-day study period. Sperm functional tests—namely a hypo-osmotic swelling test that indicates healthy viable spermatozoa (Jeyendran et al, 1984); a sperm acrosomal intactness test that indicates the functional status of the sperm acrosome and its ability to penetrate the oocyte (Gopalkrishnan, 1995); and a sperm mitochondrial activity index test that indicates motility disorders and flagellar and mitochondrial defects (Gopalkrishnan et al, 1991)—were carried out at monthly intervals to the end of the 540-day study period. A score above 60% for the hypo-osmotic swelling test and a score above 50% for the tests for acrosome intactness and the sperm mitochondrial activity index were considered normal, whereas below those levels were considered to indicate subfertility or infertility. Testicular samples were obtained by needle biopsy under sodium thiopentone anesthesia (20 mg/kg body weight) after 180, 300, 360, 390, 420, and 540 days of vas occlusion for morphological observations through light microscopy. Ultrastructural studies were carried out in the testis after 540 days of vas occlusion.

Results

The body weight of the animals did not show significant changes prior to and following vas occlusion. A slight scrotal swelling that persisted for a week was observed invariably in all the treated animals within 2 days of vas occlusion. However, the animals did not show any signs of discomfort following surgery. All animals responded to electrostimulation and ejaculated semen throughout the study period.

Semen Analysis

Physical Characteristics

The volume, ejaculation time, color, consistency, and pH of the semen did not show appreciable changes from those of pretreatment values throughout the study period. Coagulum in semen was absent throughout in 6 of the 10 vas-occluded animals.

Sperm concentration showed a drastic reduction in the first ejaculate 30 days following vas occlusion in 5 out of 10 vas-occluded animals; 2 animals were azoospermic, whereas the remaining 3 animals showed normozoospermia. In the second ejaculation 60 days following vas occlusion, 7 of 10 animals were azoospermic, and the remaining 3 animals showed oligozoospermia. Uniform azoospermia was observed from the fourth ejaculation onward, 120 days following vas occlusion, that continued until the end of the 540-day study period. The ejaculated spermatozoa were immotile, except in 1 animal (PM 09), which showed 22% motility only in the first ejaculate, and the majority of the spermatozoa were abnormal and dead (Table 1).

Table 1. . Sperm characteristics of langur monkeys prior to and following vas occlusion with SMA
  
ParameterTreatment SchedulePM 01PM 02PM 03PM 04PM 05PM 06PM 07PM 08PM 09PM 10
Sperm concentration (million/mL)Pretreatment (mean ± SE)140 ± 1.50133 ± 4.51155 ± 14.00145 ± 10.50148 ± 3.50106 ± 3.5093 ± 3.00155 ± 2.50154 ± 3.00146 ± 11.50
 Treatment          
     30 d101.2106.9121.3NilNil3341235229
     60 dNilNilNilNilNil13Nil32Nil17
     90 dNilNilNilNilNil04Nil06Nil09
     120–540 dNilNilNilNilNilNilNilNilNilNil
Sperm motility (%)Pretreatment (mean ± SE)61 ± 1.0062 ± 2.5059 ± 1.0030 ± 10.0038 ± 11.5055 ± 1.0045 ± 3.0063 ± 1.5059 ± 3.0055 ± 2.50
 Treatment          
     30 dNilNilNilNilNilNil22Nil
     60 dNilNilNil
     90 dNilNilNil
     120–540 d
Sperm viability (%)Pretreatment (mean ± SE)56 ± 1.0060 ± 1.0053 ± 2.0045 ± 5.0059 ± 1.0059 ± 1.0046 ± 1.5056 ± 1.5056 ± 1.5050 ± 1.00
 Treatment          
     30 dNilNilNil5158278
     60 dNil1311
     90 dNilNilNil
     120–540 d
Sperm abnormality (%)Pretreatment (mean ± SE)31 ± 1.0039 ± 6.5138 ± 1.0046 ± 1.0047 ± 2.5042 ± 1.5053 ± 1.5044 ± 3.0040 ± 2.0056 ± 4.00
 Treatment          
     30 d7085747972855388
     60 d918077
     90 d958897
     120–540 d

Sperm Ultrastructure

All of the ejaculated spermatozoa, since first ejaculation, observed through the scanning electron microscope were abnormal, typically head abnormalities, acrosomal disorders leading to a crater in the acrosome, midpiece damages, and coiled tail.

Sperm Functional Tests

All the sperm functional tests (ie, the hypo-osmotic swelling test, slide test for acrosome intactness, and sperm mitochondrial activity index test of the ejaculated spermatozoa) scored in the subfertile to sterile range (ie, 12% to 41% in the first ejaculate) and in the sterile range (less than 31%) in the subsequent ejaculate (Table 2).

Table 2. . Sperm functional tests of langur monkeys prior to and following vas occlusion with SMA
  
 Treatment SchedulePM 01PM 02PM 03PM 04PM 05PM 06PM 07PM 08PM 09PM 10
Mitochondrial activity index test (%)Pretreatment (mean ± SE)59 ± 1.5056 ± 4.0157 ± 2.0046 ± 1.5054 ± 4.0055 ± 4.5059 ± 3.5061 ± 1.5056 ± 2.0061 ± 1.00
 Treatment          
     30 d1218162531183821
     60 d161326
     90 d101012
     120–540 d
Acrosomal intactness test (%)Pretreatment (mean ± SE)56 ± 1.5057 ± 0.5053 ± 0.5052 ± 1.0049 ± 1.5051 ± 1.5055 ± 2.5055 ± 3.0061 ± 1.5057 ± 2.00
 Treatment          
     30 d2026232028214125
     60 d131723
     90 d080815
     120–540 d
Hypo-osmotic swelling test (%)Pretreatment (mean ± SE)57 ± 4.0160 ± 1.5060 ± 1.0057 ± 2.5052 ± 3.0056 ± 1.5060 ± 1.0056 ± 3.5157 ± 2.0059 ± 1.50
 Treatment          
     30 d1716193035233620
     60 d192831
     90 d091211
     120–540 d

Toxicological Investigations

Total red blood corpuscles (RBC); white blood corpuscles (WBC); hemoglobin (Hb); red cell indices (ie, packed cell volume [PCV], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration [MCHC]); serum protein; glucose; cholesterol; creatinine; serum glutamate pyruvate transaminase (SGPT); serum glutamate oxalate transaminase (SGOT); lactate dehydrogenase (LDH); creatine kinase (CK); bilirubin; urea; triglycerides; and HDL and LDL cholesterol levels were within the pretreatment range, although they showed wide fluctuations throughout the study period (data not shown).

Sperm Antibodies and Serum Testosterone Levels

Sperm antibody titers in serum, which ranged between 13.8 ± 0.47 and 14.3 ± 1.05 (normal values 0 to 80 U/mL), did not show statistically significant changes compared with those of pretreatment levels until 540 days of vas occlusion.

Serum testosterone levels also showed statistically insignificant changes throughout the study period (range 4.8 ± 0.60 to 5.8 ± 0.47 ng/mL).

Histology of Testis

The histology of the testes of the control animals showed the seminiferous tubules containing Sertoli cells and germ cells with active stages of spermatogenesis and the interstitium containing round or oval Leydig cells with prominent nucleus and granular cytoplasm (Figure 1A).

Figure 1.

. Histology of the testis of the langur monkey. (A) Control animal showing all stages of spermatogenesis. (B) After 300 days of vas occlusion with SMA showing normal spermatogenesis and vacuolization in a few of the Sertoli cells and normal Leydig cells. (C) After 360 days of vas occlusion, showing shrunken tubules with no definite epithelium and lumen and normal Leydig cells. (D) After 420 days of vas occlusion showing tubules of different shapes and sizes and atrophied germ cells; Leydig cells are round or mesenchymal-type. (E) After 540 days of vas occlusion, showing linear tubules with germ cell vacuolization and the lumen containing cell debris and phagocytes. Magnification 100×.

Following vas occlusion, spermatogenesis continued throughout the study period. Normal stages of spermatogenesis were observed up to 300 days of vas occlusion. The Sertoli cells and germ cells appeared normal; few of the Sertoli cells, however, showed vacuolization, and few seminiferous tubules showed sloughed germ cells in the lumen. The majority of the Leydig cells were round or oval and contained prominent nucleus and granular cytoplasm (Figure 1B).

Following 360 to 390 days of vas occlusion, the seminiferous tubules showed a different pattern. There was no definite epithelium and lumen in the seminiferous tubules. The seminiferous tubules appeared shrunken and the basal lamina of the tubules appeared thin. Germ cell differentiation, beyond the level of spermatocytes, was not observed. The lumen contained cell debris. Leydig cells were of the round, oval, or linear mesenchymal stage. The effects were focal, observed mainly in the central portion of the tubule. Few peripheral tubules also showed a degenerative pattern (Figure 1C).

Seminiferous tubules after 420 and 540 days vas occlusion were characterized by different shapes and sizes. The epithelium contained only a few germ cells with pyknotic nuclei. The lumen contained cell debris and phagocytes. The effects were focused on the central portion of the testis. However, most of the peripheral region contained normal tubules during all stages of spermatogenesis (Figure 1D and E).

Ultrastructure of the Testis

Ultrastructure of the testis of control animals showed the cellular characteristics of an active secretory state. The Sertoli cells were characterized by the indented nucleus with granular nucleoplasm and distinct nuclear membrane. The nucleolus was prominent in some of the nuclei of Sertoli cells. The cytoplasm contained numerous mitochondria, smooth and rough endoplasmic reticulae, and occasional Golgi bodies. Secretory granules and lipid droplets appeared scattered. The spermatocytes appeared round with a round or oval nucleus containing condensed chromatin and a distinct nuclear membrane. The cytoplasm contained numerous mitochondria and other granular inclusions. The nucleus of the round spermatids contained a distinct nuclear membrane and patchy chromatin material. The supranuclear cytoplasm was rich with mitochondria. Golgi bodies with numerous vesicles were prominent. Spermatids at the cap phase were distinct. The Leydig cells showed steroidogenic features with abundant smooth endoplasmic reticulum and the typical crystal of Reinke (Figure 2A through F).

Figure 2.

. Ultrastructure of the testis of control langur monkeys. (A) Sertoli cell with indented nucleus and a distinct nucleolus. The cytoplasm contains numerous mitochondria, and the nuclear membrane is distinct. Magnification 5600×. (B) The cytoplasm of the Sertoli cells showing numerous mitochondria, stacks of granular endoplasmic reticulum, tubules of smooth endoplasmic reticulum, and scattered secretory granules. Magnification 8200×. (C) The spermatocyte appears normal with an oval nucleus, condensed chromatin, and distinct nuclear membrane, and the cytoplasm is rich with mitochondria. Magnification 5600×. (D) The round spermatid at the supranuclear region showing well-defined Golgi bodies with numerous Golgi vesicles. Magnification 6200×. (E) The spermatid at “cap phase” showing the prominent acrosome cap with distinct membrane structures and rich chromatin material in the nucleoplasm. Magnification 5600×. (F) The Leydig cell showing the cytoplasm with rich tubules of smooth endoplasmic reticulum and the characteristic crystal of Reinke. Magnification 7200×. N indicates nucleus; Nu, nucleolus; M, mitochondria; Rer, granular endoplasmic reticulum; Ser, smooth endoplasmic reticulum; SG, secretary granules; GV, Golgi vesicles; R, crystal of Reinke.

Following 540 days of vas occlusion, the ultrastructure of the testis, particularly of the affected tubules, confirmed the histological findings. The cytoplasm of the Sertoli cells showed vacuolization. The nucleus of the Sertoli cells, however, appeared normal. Although mitochondria, smooth and rough endoplasmic reticulae, Golgi bodies, and secretory granules were found in the cytoplasm of the Sertoli cells, the mitochondria and the Golgi bodies showed degenerative features. Vacuolization was also observed in the cytoplasm of spermatids, and their nuclear membrane showed degeneration. Leydig cells appeared normal with an abundance of smooth endoplasmic reticulum, mitochondria, and secretory granules (Figure 3A through D). However, the seminiferous tubules with normal spermatogenesis showed ultrastructural features similar to those of control.

Figure 3.

. Ultrastructure of the testis of the langur monkey after 540 days of vas occlusion with SMA. (A) The Sertoli cell showing the indented nucleus with moderate chromatin network; the cytoplasm shows vacuolization and the cell organelles show degenerative features. Magnification 5600×. (B) The round spermatid showing the nucleus with degenerative features (arrow). Magnification 5600×. (C) The spermatid at the Golgi phase showing the nucleus with membrane disorders (arrow); the acrosome cap shows vacuolization (double arrows), and Golgi bodies are prominent. Magnification 5600×. (D) The Leydig cell showing the cytoplasm with abundant smooth endoplasmic reticulum, well-defined mitochondria, Golgi bodies, and secretory granules. Magnification 8400×. G indicates Golgi bodies; Ser, smooth endoplasmic reticulum; M, mitochondria; SG, secretory granules.

Discussion

In the present investigation it has been observed that the semenological pattern in the initial ejaculations following vas occlusion with SMA is almost similar to that of short-term vas occlusion for 150 days in langur monkeys (Lohiya et al, 1998a,b). Azoospermia resulted after 120 days of vas occlusion and continued until the end of the 540-day study period. Spermatozoa of the initial ejaculations were immotile with damages in the acrosome and midpiece, indicating that the spermatozoa are incapable of penetrating the eggs. The normal serum testosterone levels, hematology, and serum clinical parameters throughout the course of investigations suggest the safety of the procedure clinically as well as at the level of accessory reproductive glands. Absence of sperm antibodies in the serum up to 540 days of vas occlusion is considered to be more advantageous since sperm antibodies would interfere with the functional success of occlusion reversal (Alexander, 1977; Flickinger et al, 1995).

It has been reported that in a vasectomy the resultant damage to spermatogenesis is mainly due to an increase of pressure in the seminiferous tubules and autoimmune reaction. The consequences of vasectomy have been related to germ cell death by apoptosis; thickening of seminiferous tubules, particularly the collagen layer between the basal lamina of the seminiferous epithelium and the myoid cells; and increased leukocyte infiltration in the boundary zones of degenerated seminiferous tubules (Lue et al, 1997; Shen et al, 1998; Dobson et al, 2000; Whyte et al, 2000). Pressure-mediated damage to the seminiferous epithelium reported to be followed by sperm granuloma formation and obstruction in the epididymal head (McDonald, 2000). Ultrastructural study indicated that the spermatogonia and Sertoli cells are the most resistant cells to vasectomy and are even observed in some regenerating testes lacking a complete germinal epithelium. Interstitial cells, however, remained unaffected (Whyte et al, 2000).

Structural changes in the seminiferous epithelium following vasectomy in dogs showed greater extracellular spaces and the appearance of immature germinal cells and multinucleated spermatids in all stages of spermatogenesis. A relative increase in the size of Sertoli cells as well as their phagocytic function without any changes in the Leydig cells have also been reported (Whyte et al, 1999). Intraepithelial vesicle formation, loss of germ cells, intraluminal macrophages, and lymphocytic infiltration following vasectomy have been reported in guinea pigs (Aitken et al, 1999). Immunohistochemical and ultrastructural studies revealed that the process of spermatogenesis deteriorates more severely in testes with dense IgG deposition (Aydos et al, 1998a). It has also been suggested that the overproduction of reactive oxygen species (ROS) following vasectomy could induce testicular damage, since malonaldehyde, a principal marker of lipid peroxidation, shows a positive correlation to testicular damages in vasectomized animals (Aydos et al, 1998b).

In the present investigation, the testes histology following 540 days of vas occlusion resulted in bilateral focal degeneration, showing disorganization of the Sertoli cells and germ cells with evidence of phagocytosis in the lumen. Ultrastructural features indicated that these cells are most susceptible to vas occlusion, leading to vacuolization in many of the seminiferous tubules. The vesicle formation, germ cell atrophy, and intraluminal macrophages were similar to the observations made in vasectomized guinea pigs (Aitken et al, 1999). The pressure damage to the seminiferous epithelium, as has been reported, is followed by sperm granuloma formation and obstruction in the epididymal head (McDonald, 2000). In the present investigation, the observed damage to the seminiferous epithelium after 360 days of vas occlusion—together with the fact that there is no granuloma formation up to the end of the 540-day study period and the cauda epididymal epithelium appeared normal throughout the study period (unpublished observations)—suggest less feasibility of pressure-mediated damage. The testicular damage could, however, be related to the overproduction of reactive oxygen species due to increased intraluminal macrophages and lymphocytic infiltration, which would normally increase ROS production (Aitken et al, 1999). The focal degeneration as observed in the present investigation could be more advantageous, as the spermatogenesis was continuous, which becomes more favorable when there is a need for reversal. Another advantage of this procedure is that there was no sperm granuloma formation during the 540-day study period, reducing the risk of sperm antibodies.

Acknowledgement

Ultrastructural studies were carried out at the Regional Centre for Sophisticated Instrument Facility for Electron Microscopy in the Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi. The authors are thankful to Dr Sujoy K. Guha, Professor of Biomedical Engineering, Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi, India, for providing SMA.

Footnotes

  1. Supported by the Ministry of Health and Family Welfare, Government of India, New Delhi.

Ancillary