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- Complementary Medicine -
Sperm motility inhibitory effect of the benzene chromatographic
fraction of the chloroform extract of the seeds of
Carica papaya in langur monkey, Presbytis entellus
entellus
Nirmal K. Lohiya, Boomi Manivannan, Shipra Goyal, Abdul S. Ansari
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur 302004, India
Abstract
Aim: To assess the contraceptive efficacy of the benzene chromatographic fraction of the chloroform extract of the
seeds of Carica papaya in langur monkeys.
Methods: The test substance was given p.o. to five monkeys at 50 mg/kg
body weight/day for 360 days. Control animals
(n = 3) received olive oil as vehicle. Sperm parameters as per World
Health Organization standards, sperm functional tests, morphology of testis and epididymis, haematology, clinical
biochemistry, serum testosterone and libido were evaluated. Following completion of 360 days treatment the animals
were withdrawn from the treatment and the recovery pattern was assessed by semen analysis and sperm functional
tests. Results: Total inhibition of sperm motility was observed following 60 days of treatment that continued until
360 days study period. Sperm count, percent viability and percent normal spermatozoa showed a drastic decline
following 30 days of treatment. Sperm morphology showed predominant mid piece abnormalities. Sperm functional
tests scored in sterile range. Histology and ultrastructure of testis revealed vacuolization in the Sertoli cells and germ
cells. Loss of cytoplasmic organelles was evident in spermatocytes and round spermatids. Histology and
ultrastructure of epididymis of treated animals were comparable to those of control animals. Hematological and serum clinical
parameters and testosterone levels fluctuated within the control range throughout the study period. Recovery was
evident following 60_120 days of treatment withdrawal.
Conclusion: The results suggest that the benzene
chromatographic fraction of the chloroform extract of the seeds of
Carica papaya shows contraceptive efficacy without
adverse toxicity, mediated through inhibition of sperm motility.
(Asian J Androl 2008 Mar; 10: 298_306)
Keywords: male contraception; Carica papaya
seeds; sperm motility inhibition; testis; epididymis; langur monkeys
Correspondence to: Prof. Nirmal K. Lohiya, Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur,
302004, India.
Tel: +91-141-270-1809, +91-141-270-3552
Fax : +91-141-270-1809
E-mail: lohiyank@hotmail.com, lohiyank@rediffmail.com
Received 2007-01-29 Accepted 2007-07-23
DOI: 10.1111/j.1745-7262.2008.00331.x
1 Introduction
The contraceptive efficacy of the seeds of Carica
papaya at various level of extraction/purification has been well
established in rats, rabbits and langur monkeys [1]. In our earlier preliminary investigations, aqueous, methanol,
ethanol, ethyl acetate and chloroform extracts of the seeds of
Carica papaya have been shown to produce 100%
sterility in rats. In rabbits, aqueous extract was ineffective and chloroform extract produced azoo-spermia after
120 days of treatment [1]. In langur monkeys, treatment with chloroform extract resulted in azoospermia after
90 days of treatment [2]. Among the various crude extracts tested, the chloroform extract was found to be more
effective in eliciting contraceptive efficacy in rats, rabbits and langur monkeys. We have attempted to purify the
chloroform extract in column chromatography eluted with
benzene. The resultant benzene chromatographic
fraction was further purified by solvent subfractionation
using methanol and ethyl acetate. At each stage of
purification, the purified fractions were tested for its
contraceptive efficacy in animal models (i.e. rats,
rabbits and langur monkeys) to establish, if there are losses
of biological activity of the fractions in purification
process. Initial attempts to identify the chemical
composition of the purified fractions through high
performance liquid chromatography (HPLC), nuclear magnetic
resonance-1 (NMR-1), and mass spectroscopy (MS)
revealed that the product is a homogenous mixture of fatty
esters (unpublished observations). However, in the
partial purified form, these fractions showed no loss of
biological activity, yielding total sperm motility inhibitory
effect in rats [1, 3] and azoospermia in rabbits [4]. It is
also pertinent to mention that column chromatography
of chloroform extract eluted with methanol or ethyl
acetate in place of benzene, did not produce significant
contraceptive efficacy in either rats [1] or rabbits [4]. All
these effects were free of toxicity, evidenced from
routine hematology and clinical chemistry and reversible
following withdrawal of treatment [1].
Although there are established species specificity, i.e.
sperm motility inhibition in rats and azoospermia in rabbits,
owing to the proven contraceptive efficacy of the
benzene chromatographic fractions resulting in 100% sterilit
y
in these animal models, a further insight of the study in a
non-human primate model is warranted to extrapolate the
findings to human. Therefore, in the present investigation,
an attempt was made to assess the contraceptive efficacy,
toxicological profiles and mode of action at tissue level
of benzene chromatographic fraction of the chloroform
extract in langur monkeys, an animal model close to
human in reproductive anatomy and physiology [5].
2 Materials and methods
2.1 Test material
Fresh seeds of Carica papaya of honey dew variety
were used in the present investigation after
authentication in the Department of Botany, University of Rajasthan,
Jaipur, India (voucher No. RUBL 16590). The seed
extracts were prepared as per the standarized protocol [3]
for oral administration to langur monkeys.
2.2 Animals
Ten adult male langur monkeys Presbytis entellus
entellus Dufresne, weighing 15 kg_18 kg [5] were
procured from places in and around Jaipur, India and were
kept for quarantine in individual metallic cages (105 cm
× 75 cm × 75 cm) in the Departmental Primate House
Facility for a period of 2 months. The animals were fed
with roasted wheat cakes, seasonal vegetables and fruits,
and water was provided ad libitum. Routine
pathological tests and semen analysis were carried out to assess
the health and reproductive status and only healthy
animals (n = 8) were selected for the investigation. The
experiments were conducted in accordance with accepted
humane practices as approved by the Departmental
Research/Ethical Committee and the Guidelines for Care
and Use of Animals for Scientific Research [6] were
strictly followed. Complete veterinary care and
supervision were provided to the animals throughout the course
of the investigation.
2.3 Experimental design
2.3.1 Pretreatment phase
Prior to the onset of the experiments, at least three
pretreatment semen and blood samples at 10-day
intervals were collected and subjected to routine analysis of
semen, hormone, hematology and serum clinical biochemistry, and the values were used as individual
reference control for comparison.
2.3.2 Treatment phase
Five animals were treated with the benzene chromatographic fraction of the seeds of
Carica papaya using a sterile feeding
syringe at the standardized dose regimen, 50 mg/kg body weight/day p.o. for 360 days
[2]. Three animals served as vehicle treated control.
The animals were habitual for the dosage and did not
show dislike/discomfort during treatment.
2.3.3 Recovery phase
Following completion of 360 days treatment, all the
animals were withdrawn from the drug treatment. The
pattern of recovery was assessed through semen
analysis and sperm functional tests.
2.4 Parameters
The following parameters were analyzed:
2.4.1 Body weight
Body weight of the animals before, during the
treatment and recovery periods was recorded monthly.
2.4.2 Semen analysis
Semen samples were collected every 10 days before
treatment and every 30 days during treatment and
recovery periods by penile electro stimulation [5] for the
following analyses:
2.4.2.1 Physical characteristics
Semen volume, ejaculation time, pH value, colour,
consistency, sperm concentration, motility, viability and
morphology were analyzed according to the World Health
Organization Method Manual [7].
2.4.2.2 Scanning electron microscopy
Washed spermatozoa were immediately fixed in
2.5% glutaraldehyde in phosphate buffer for 30 min and
washed thrice in phosphate buffer (0.1 mol/L; pH 7.2).
A thin film of spermatozoa was smeared on a scanning
electron microscopy stub with silver paint, air dried,
sputter coated with gold and observed under a scanning
electron microscope (Leo 435 VP, Eindhoven, the Netherlands).
2.4.2.3 Sperm functional tests
The acrosome intactness test for the status of acrosome [8], the sperm mitochondrial activity index
test, indicating motility disorders, flagellar and
mitochondrial defects [9] and hypo-osmotic swelling test,
indicating the membrane integrity [10], were carried
out using washed spermatozoa. Scores below 50% in
the acrosome intactness and mitochondrial activity
index tests and below 60% in the hypo-osmotic swelling
test were considered infertile.
2.4.3 Histology and ultrastructure
2.4.3.1 Light microscopy
Following completion of the treatment schedule and
recovery period, the animals were anaesthetized with
sodium thiopentone (20 mg/kg body weight, i.v.).
Biopsies were obtained from testis and epididymis and a
portion of the tissues were fixed immediately in Bouin's fluid
for 24 h, dehydrated in ethanol, cleared in xylene and
embedded in paraffin wax. Five micron thick sections
were double stained with Harris' hematoxylin and eosin
for observation.
2.4.3.2 Electron microscopy
For ultrastructural studies, the remaining biopsies of
testis and cauda epididymis were cut into small pieces
(1 mm) and fixed immediately in 2.5% glutaraldehyde in
phosphate buffer (0.1 mol/L; pH 7.2) for 24 h. After
primary fixation, the tissues were washed thoroughly in
phosphate buffer, post fixed in 1% OsO4 for 6 h, washed
in phosphate buffer, followed by distilled water,
dehydrated in acetone, infiltrated and embedded in low
viscosity spurr media and polymerized at 60ºC for 48 h.
The semithin sections of 1 mm were stained with
toluidine blue and ultrathin sections were stained with uranyl
acetate and lead citrate and observed under transmission
electron microscope (Philips CM-10, Eindhoven, the
Netherlands).
2.4.4 Toxicological investigations
2.4.4.1 Hematology
Total red blood corpuscles (RBC), white blood
corpuscles (WBC), hemoglobin and standard hematological
indices (i.e. packed cell volume [PCV], mean
corpuscular volume [MCV], mean corpuscular hemoglobin
[MCH], mean corpuscular hemoglobin concentration [MCHC]) were recorded monthly [11].
2.4.4.2 Clinical biochemistry
Serum total protein, glucose, cholesterol, creatinine,
creatine kinase (CK), serum aspartate aminotransferase
(S AST), serum alanine aminotransferase (S ALT) were
estimated through Autoanalyzer (Erba Smartlab, Mumbai,
India) using reagent kits (Transasia Biomedicals, Mumbai,
India).
2.4.5 Hormone analysis
Serum testosterone was assayed quarterly by enzyme
immunoassay using reagent kits (Biochem Immuno Systems, Italia, Italy) and the values were recorded
using a microplate reader (Tecan, Grödig, Austria).
2.4.6 Libido
Libido was assessed monthly through response to
electro-stimulation and mounting behavior [5].
2.5 Statistical analysis
Whenever appropriate, paired t-test was used for
statistical comparison. The values are expressed as mean ±
SD and P < 0.05 was considered
significant. Data were analyzed using SPSS version 10.0 software
(SPSS, Chicago, IL, USA).
3 Results
3.1 Body weight
The body weight of the treated animals did not show
significant changes compared to that of control animals
throughout the study period (data not shown).
3.2 Semen analysis
3.2.1 Physical characteristics
The ejaculation time, volume, pH, color, consistency,
liquefaction time and liquefaction status did not show
significant changes compared to pretreatment values in both
control and treated animals throughout the study period.
3.2.2 Sperm concentration
A gradual decline in sperm count compared to
pretreatment/control values was recorded from 30 days of
treatment onwards and at 360 days study period, sperm
count ranged between 70 × 106/mL and 80 ×
106/mL. The treatment withdrawal resulted in a gradual increase
in sperm count from 30 days. Sperm count comparable
to that of the pretreatment values was achieved
following 120_150 days of treatment withdrawal. Control
animals showed normal sperm concentration throughout the
study period (Figure 1A).
3.2.3 Sperm motility
A drastic reduction, compared to
pretreatment/control values in percent sperm motility ranged between 19%
and 26% was observed within 30 days of treatment. A
total and uniform inhibition of sperm motility was
observed after 60 days of treatment that continued until
360 days study period. Following treatment withdrawal,
sperm motility resumed after 30 days; however, with
teratozoospermia, motility increased gradually and attained
pretreatment levels after 120 days of treatment
withdrawal. Control animals exhibited normal sperm motility
throughout the study period (Figure 1B).
3.2.4 Sperm viability
The percent viable spermatozoa showed a drastic
decline (range 10%_30%) from 30 days of treatment
compared to control/pretreatment values, which
continued throughout the 360 days study period, indicating
negative fertility. Treatment withdrawal resulted in a
gradual improvement in percent sperm viability. The
pretreatment levels were achieved after 90 days of
treatment withdrawal. Control animals exhibited normal sperm
viability throughout the study period (Figure 2A).
3.2.5 Sperm abnormality
The percent abnormal spermatozoa showed a steep
increase compared to control/pretreatment values from
30 days treatment period onwards (range 68%_72%),
which continued throughout the 360 day treatment
period. Following treatment withdrawal, the percent
abnormal spermatozoa resumed to pretreatment level
within 30_60 days. Control animals did not show drastic
changes from their pretreatment values throughout the
study period (Figure 2B).
3.3 Scanning electron microscopy of spermatozoa
Scanning electron microscopy of the spermatozoa
showed abnormalities mainly at mid-piece and neck
regions from 30 days of treatment. Coiled tail was also
frequently observed (Figure 3). Treatment withdrawal
resulted in normal morphology after 90 days.
3.4 Sperm functional tests
All sperm functional tests scored in sterile range from
30 days of treatment, which fluctuated within the sterile
range throughout the 360-day study period. Treatment
withdrawal resulted in a gradual increase and the normal
parameters showing the fertile score, comparable to the
pretreatment level were achieved after 90 to 120 days of
treatment withdrawal. All sperm functional tests in
control animals scored in the fertile range throughout the
study period (Figure 4).
3.5 Histology and ultrastructure
3.5.1 Morphology of testis
3.5.1.1 Light microscopy
Following completion of the 360-day treatment period, testis histology revealed affected tubules.
Although in most of the seminiferous tubules all stages of
spermatogenesis were present, at certain places the
spermiogenesis was arrested at stage I of the spermatogenic
cycle. Vacuolization in Sertoli cells and germ cells were
evident in the majority of the seminiferous tubules.
Sertoli cells and round spermatids were vulnerable to
vacuolization. Few of the spermatocytes showed
evidence of nuclear pyknosis. Leydig cells, however,
appeared normal (Figure 5).
3.5.1.2 Electron microscopy
Ultrastructure of the testis of the treated animals,
following completion of the 360-day treatment period,
showed increased vacuolization in the Sertoli cells and
germ cells compared to the control. The nucleus of
the Sertoli cell appeared normal with deep indentation
and dense chromatin material and the entire cytoplasm
showed vacuolization and loss of cytoplasmic organelles. Mitochondria were few and vacuolated.
Nuclear degeneration and loss of cytoplasmic organelles
were evident in spermatocytes and round spermatids
and the nuclear membrane of these cells were ill-defined.
Vacuolization in the acrosomal cap and peripheral
mitochondria of round spermatids was also evident
(Figures 6_8).
3.5.2 Morphology of epididymis
3.5.2.1 Light microscopy
Histology of the cauda epididymis following 360 days
treatment with benzene chromatographic fraction
presented no appreciable changes to that of the control
animals. The basal and principal cells appeared normal
and comparable to that of control animals. Lumen
contained dense spermatozoa. However, few phagocytes
were evident in the pool of spermatozoa. Inter-tubular
elements appeared normal (figure not shown).
3.5.2.2 Electron microscopy
Ultrastructure of the principal cells of cauda
epididymis following 360 days of treatment was comparable to
that of the control. The nucleus was round with
prominent double layered nuclear membrane and the
nucleoplasm contained dense chromatin material.
Occasionally the nucleus showed an irregular pattern.
Mitochondria in the cytoplasm were round or elongated with
prominent cristae. Supranuclear cytoplasm was characterized
by the presence of numerous well-defined Golgi bodies,
tubules of granular endoplasmic reticulum and rosettes
of glycogen granules, showing evidence of active
protein secretion (figure not shown).
3.6 Toxicological investigations
There were no appreciable changes, although wide
fluctuations were observed in the levels of total RBC,
WBC, hemoglobin (Hb), PCV, MCV, MCH and MCHC and the serum protein, glucose, cholesterol, creatinine,
CK, serum glutamate pyruvate transaminase (SGPT) and
serum glutamate oxalate transaminase (SGOT). The values fluctuated within the pretreatment range in both
control and treated animals throughout the study period
(data not shown).
3.7 Hormone assay
Serum testosterone levels did not show appreciable
changes from that of the pretreatment values in both
control and treated animals throughout the study period
(Figure 9).
3.8 Libido
The libido of the treated animals remained unaffected.
All the animals, control and treated, showed normal
mounting and ejaculatory behavior throughout the study
period.
4 Discussion
The seed extracts of Carica papaya produce
variable responses towards contraceptive efficacy in rats,
rabbits and langur monkeys. The effects are post-testicular, affecting the cauda epididymal sperm motility
in rats, and testicular effects lead to azoospermia in
rabbits and langur monkeys [1_4]. In the present investigation,
treatment with benzene chromatographic fraction of the
chloroform extract of the seeds of Carica
papaya resulted in decline in sperm concentration after 30 days of
treatment and total sperm motility inhibition after 60 days
of treatment with reduced viability and increased sperm
abnormality. Sperm functional tests including
ultrastructure of spermatozoa, indicated that these spermatozoa
are functionally infertile, suggesting significant
contraceptive efficacy of the test material.
The mechanism by which the extract of the seeds of
Carica papaya induces the sperm motility inhibition in all
treated animals in the present investigation is poorly
understood. It has been reported that there are three
major sites of action of an epididymal antifertility agent;
namely, (i) the peritubular muscle, hastening sperm
transport leading to ejaculation; (ii) on the epithelium by
altering the composition of epididymal fluid and (iii) on the
spermatozoa by affecting their enzymes [12]. As there
are no appreciable changes in the histology and
ultrastructure of epididymis in the treated animals, it is likely
that the effect lies directly on the epididymal
spermatozoa [13].
The excessive generation of reactive oxygen species
might result into dramatic loss of sperm function and
motility [14]. Decrease in activity of antioxidant enzymes
in the epididymis increase reactive oxygen could damage
the epididymal environment and sperm function [15].
Al-Majed et al. [16] demonstrated that treatment with
Yohimbe induced chromosomal aberrations, spermatozoal abnormalities and reduction in sperm count, motility
and fertility in Swiss albino mice as a result of depletion
of antioxidants. Likewise, in the present study, the testis
histology and ultrastructure depicted inhibition of
spermatogenesis and an increased percentage of abnormal
spermatozoa.
In the triptolide treated rats, Huynh
et al. [17] described two phenotypic effects on mature and maturing
germ cells. The first action appears early on the
epididymal sperm and its subsequent action is directly on the
germ cells of testis. Interestingly, in the triptolide-treated
rats, no ultrastructural differences in the epididymal
epithelium were observed between control and treated rats,
but the treated rats exhibited total motility inhibition with
severe sperm abnormalities and varied sperm
concentration in the cauda epididymis [17]. In the present
investigation, the treatment with the seed products of
Carica papaya altered testis histology but not that of
epididymis. Even the ultrastructure of epididymis showed
normal configuration with active protein synthetic
machinery, which provides a clue that the test substance
directly acted upon the germ cells of the testis,
particularly at the level of spermatid maturation and produced
malformed spermatozoa, resulting in increased
abnormalities with motility inhibition of spermatozoa in the
ejaculation.
It has been reported that the Sertoli cells of a
regressed testis do not contain conspicuous stacks of
granular endoplasmic reticulum, which is consistent with
a reduced protein secretory function of the Sertoli cells
[18]. Huynh et al. [17] reported ultrastructural defects
in the Sertoli cells and spermatids of the triptolide treated
rats. In the present investigation, treatment with a
benzene chromatographic fraction of the chloroform extract
of the seeds of Carica papaya showed vacuolization in
the Sertoli cell cytoplasm and loss of cytoplasmic
organelles, particularly at the basal region, suggesting loss
of metabolic activity of the cell. Degeneration and
maturational arrest of germ cells (i.e. spermatocytes and
spermatids), therefore, could be due to the Sertoli cell
factors responsible for germ cell maturation. It has also
been described that gossypol and gossypol acetic acid
produce vacuolization and degeneration of mitochondria
in the mid-pieces of late spermatids in rats and hamsters
that are pathognomonic to gossypol or its derivatives [19].
A similar unique effect on the mitochondria of late
spermatids and on the voided spermatozoa has been observed
in monkeys treated with chloroform extract of the seeds
of Carica papaya [2]. In the present study, the sperm
functional tests (i.e. acrosome intactness test for the
acrosome status and the ability of the spermatozoa to
penetrate the oocyte, sperm mitochondrial activity
index test for motility disorders and flagellar and
mitochondrial defects in the mid-piece and the hypo-osmotic
swelling test for membrane integrity [8_10]),
particularly the tests for mitochondria and membrane integrity,
indicated defective sperm production from the testis [16].
Incidentally, in the present study, ultrastructure of the
spermatocytes and spermatids showed nuclear degeneration and vacuolized mitochondria.
It is concluded here that the sperm motility
inhibitory effect could be a result of a selective action of the
test substance on developing germ cells, possibly
mediated through Sertoli cells, leading to inhibition of
mitochondrial activity of these cells, which might alter the
respiratory chain, generating a cytotoxic effect on the
germ cell proliferation, as in the case of gossypol treated
animals [19, 20]. It is further evidenced by increased
mid-piece defects and vacuolization in the mitochondria
in the voided spermatozoa of the seed extract-treated
animals in the present study, as reflected from sperm
ultrastructure. However, further evidence needs to be
generated at the level of glycolytic pathway of the testis,
more particularly on the developing germ cells and
spermatozoa of the treated animals to substantiate this view.
The blood parameters (i.e. RBC, WBC, Hb, standard hematological indices, the serum clinical parameters and
the serum testosterone level, which presented normal
values throughout the study period) suggest systemic
safety of the test material.
Available evidence indicates that the benzene
chromatographic fraction of the chloroform extract of the
seeds of Carica papaya possesses contraceptive efficacy,
effected through defective germ cell proliferations, as
evidenced from testis ultrastructure. The sperm motility
inhibitory action in the present study seems to be more
advantageous in that it would avoid delayed reversibility
as in the case of gossypol treated animals [20]. The
results achieved so far meet the essential criteria for the
male contraceptive (i.e. being orally effective,
non-steroidal, free of toxicity, reversible and possibly cost
effective). However, further purification of the
chromatographic fraction, identification of bioactive
compounds, their characterization and pharmacokinetic
properties of the purified compounds are required for
product development and mass application. Further
relevant studies are currently in progress.
Acknowledgment
The investigations were supported by the Ministry
of Health and Family Welfare/Indian Council of Medical
Research, Government of India, New Delhi and the
Special Assistance Programme (Phase III), University Grants
Commission, New Delhi. Ultrastructural studies were
carried out at EM-SAIF-DST Facility, Department of
Anatomy, All India Institute of Medical Sciences, New
Delhi, India.
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