JBRA Assist. Reprod. 2021;25(1):97-103
ORIGINAL ARTICLE
doi: 10.5935/1518-0557.20200058
CREM, PRM I and II gene expression in Wistar rats testes treated with antipsychotic drugs: Chlorpromazine, Rauwolfia vomitoria and co-administration of reserpine, zinc and ascorbic acid
1Department of Anatomy, College of Health Sciences, Osun State University, Osogbo, Osun State, Nigeria)
2Department of Anatomy, College of Health Sciences, University of Ilorin, Kwara State, Nigeria
3Department of Anatomy, College of Medicine and Health Sciences, Ekiti State University, Ekiti State, Nigeria
4Department of Anatomy, University of Medical Sciences, Ondo City, Ondo State, Nigeria
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
ABSTRACT
Objective: The literature has shown that synthetic antipsychotic drugs induce
reproductive toxicity, while psychiatric patients treated with traditionally
used antipsychotic herbs (Rauwolfia vomitoria) showed no
traces of reproductive toxicity. Thus, this study aimed to investigate the
expression of CREM, PRM I and II genes in the testes of Wistar rats treated
with antipsychotic drugs: chlorpromazine, Rauwolfia
vomitoria (RV) and co-administration of
reserpine, zinc and ascorbate (RAZ).
Methods: Forty-five adult male Wistar rats with rats with average weight of
180±4.67g were divided into nine groups (A-I) (n=5). Group A was
administered saline (control) while rats in Groups B and C received 10 and
20mg/kg body weight (bwt) of chlorpromazine respectively. Groups D and E
received 2.5 and 5mg/kg bwt of reserpine, respectively; while Groups F and G
received 150 and 300mg/kg bwt of RV leaf extract. Groups H
and I received (2.5+5+100) mg/kg bwt and (5+10+200) mg/kg of combination of
RAZ, respectively for 56 days.
Results: The CREM, PRM I and II genes were significantly downregulated while
significant decreased in serum FSH and testosterone concentration were found
in the Chlorpromazine- and Reserpine-treated groups. Groups H and I showed a
highly significant upregulation of the CREM, PRM I and II genes, and a
highly significant increase in serum FSH and testosterone
concentrations.
Conclusion: The study concluded that the HPT-Axis was impaired by chlorpromazine and
reserpine, while RV and a combination of RAZ administration
enhanced the axis in an animal model. The study recommended that synthetic
antipsychotic drugs should be taken with Zinc and Ascorbate in order to help
prevent reproductive toxicity associated with antipsychotic drugs. We need
further studies in humans to confirm these findings.
Keywords: antipsychotics, testes, gene expression, Rauwolfia vomitoria
INTRODUCTION
Infertility is a global health problem, and it is one of the most stressful
conditions affecting married couples. Even though not lethal, it has been described
as a radical life changing problem that carries with it significant psychological
trauma (Oyewopo et al., 2018). Infertility can be caused by various problems in which synthetic
antipsychotic drugs are not exempted (Ali &
Bhagya, 2011).
Synthetic antipsychotic drugs are chemically synthetized drugs, used in the
management of Psychosis. Psychosis is a severe mental disorder, with physical damage
to the brain, marked by a deranged personality and a distorted view of reality or an
abnormal condition of the mind that involves a loss of contact with reality (Freudenreich, 2012). About 450 million people
suffer from mental disorders and one person in four will develop one or more mental
or behavioral disorders during their lifetime (WHO,
2003).
Many antipsychotic drugs have been developed over time to combat psychosis.
Chlorpromazine (CPZ) is one of the first generation synthetic antipsychotic drugs,
and still remains one of the most common drugs used for psychosis treatment
worldwide (Chong et al., 2004). Chlorpromazine is a dopamine antagonist of the typical antipsychotic
class of medications, possessing additional antiadrenergic, antiserotonergic,
anticholinergic and antihistaminergic properties (Healy, 2004). There are many literature reviews reporting that the
majority of psychotic patients under Chlorpromazine treatment developed infertility
problems, such as diminished libido, erectile dysfunction, impotence, ejaculation
inhibition, significant increase in serum prolactin and progesterone, significant
decrease in estradiol, testosterone and Luteinizing hormone (Zamani et al., 2015). In an animal model, Raji et al. (2005) reported that Chlorpromazine altered the activity of some androgen-dependent
enzymes, decreased the weight of rat testes, caput and cauda epididymis and
suppressed testicular functions. The Electronic Medicines Compendium (EMC) reported
that there is paucity of information as regards to anti-fertility effects of
Chlorpromazine in male animals when compared with female animals, where it has been
revealed to decrease fertility parameters (EMC,
2019). Ali & Bhagya (2011)
reported that Chlorpromazine results in loss of follicle by atresia and delays the
onset of puberty in immature female rats.
The search for alternative therapies for the treatment of common diseases has moved
man closer to natural products that are easy to assess and affordable. Use of these
natural products cannot be overlooked in the treatment of mental and other ailments
in Nigeria (Akpanabiatu et al., 2006). Rauwolfia vomitoria is one of the natural
products used traditionally for the treatment of mental disorders in Nigeria (Akpanabiatu et al., 2006).
Rauwolfia vomitoria (RV) belongs to the Apocynaceae
family. It is mostly found in the forest part of southern Nigeria. The plant is also
called swizzle stick in English and is about 8m in height (Orwa et al., 2009). The active phytochemical
components, as reported by Akpanabiatu et
al. (2006), are alkaloids, rauwolfine, rescinnamine,
serpentine, ajmaline serpentinine, steroid-serposterol, saponin and reserpine - an
active compound used in the treatment of psychosis. Similarly, Rauwolfia
vomitoria leaves extract have been reported to contain high
concentrations of Zinc and Vitamin C (Ascorbic acid), elements which are essential
to promote male fertility (Ogunlesi et
al., 2009). The crude extract of Rauwolfia
vomitoria has been reported to help in treating swellings in male
reproductive organs related to infertility (Sinclair, 2000). It has also been reported to have anti-prostate cancer
and anti-diabetic activities, which may serve as fertility enhancers, where
infertility is associated with such disorders (Sinclair, 2000).
Nur & Adam (2016) reported that reserpine
was one of the first potent antipsychotic drugs isolated from the Rauwolfia
vomitoria plant. Reserpine mediate depletion of monoamine
neurotransmitters in the synapses, which improves antipsychotic behaviors because of
its effect, which lasts longer than any other antipsychotic agent (Baumeister et al., 2003).
Although, reserpine used only as an antipsychotic drug has been reported to pose
some anti-pyramidal side effects (Nur & Adams,
2016). Khazan et al. (1960) reported severe atrophy and impaired spermatogenesis in the testes
of pigeons treated with reserpine, while Mosad et al. (2000) reported mild to severe degenerative
changes in rat testis after acute reserpine administration.
The literature has shown that antipsychotic drugs induce reproductive toxicity, but
there is dearth of information as regards to the molecular mechanism behind this
reproductive toxicity. Moreover, to the best of our knowledge, no study has
investigated the effects of the concurrent administration of Reserpine, Ascorbic
acid and Zinc (RAZ), the selected phytochemicals present in Rauwolfia
vomitoria leaves on male reproductive parameters. Thus, this study
aimed at assessing the impacts of Chlorpromazine, Rauwolfia
vomitoria and the co-administration of Reserpine, Ascorbic acid and
Zinc (RAZ) on the expression of CREM, PRM I and II genes in the testes of adult
Wistar rats.
MATERIALS AND METHODS
Compounds procurement and preparation
All compounds used (Chlorpromazine, Reserpine, Zinc and Vitamin C) were pure
compounds procured from Hefei TDJ Chemical Co. Ltd China and authenticated at
the Pharmacy Department - University of Ilorin, Kwara State. We dissolved 100mg
of these compounds in 100ml of distilled water: i.e. 1ml of the solution
contains 1mg of the solvent (Stock solution). We let the solution stand for some
minutes, constantly shaken for proper dissolution.
Rauwolfia vomitoria leaves authentication and Ethanolic extraction
Rauwolfia vomitoria leaves were locally collected from a
farmland in Osogbo and identified in the Division of Botany, Department of
Biological Sciences, University of Ilorin, Nigeria. Rauwolfia
vomitoria leaves were air-dried, the dried pieces were then
pulverized using an electric blender (Blender /Miller III, model MS-223, Taiwan,
China), and the extraction procedure was done as described by Adeleke et al. (2017).
Experimental Design
Forty-five adult male Wistar rats with an average weight of 180±4.67g were
divided into nine groups (A-I) (n=5). Group A was administered saline (control),
while rats in Groups B and C received 10 and 20 mg/kg body weight (bwt) of
chlorpromazine respectively. Groups D and E received 2.5 and 5 mg/kg bwt of
reserpine, respectively; while Groups F and G received 150 and 300 mg/kg bwt of RV leaf extract. Groups H and I received (2.5+5+100) mg/kg
bwt and (5+10+200) mg/kg of combination of RAZ, respectively for 56 days. Drug
administrations were done orally using an orogastric cannula. The rats were fed
with pelletized grower mash procured from Mosodun Feeds Nigeria Ltd. Osogbo,
with ad libitum access to drinking water. Ethics approval number
(UERC/ASN/2017/1067) was gotten from the University of Ilorin ethical review
committee (UERC). All procedures were done according to global best practices
and institutional guidelines on the care and use of animals.
Animal Slaughter and Sample Collection
The rats were slaughtered on the 57th day of the experiment,
anesthetized with 80mg/kg of ketamine hydrochloride. Blood was withdrawn from
the heart apex (left ventricle) for hormonal analysis, while small testicular
tissues were excised for Reverse transcriptase Polymerase chain reaction
(RT-PCR) analysis.
Hormone Measuring Assay
Serum levels of Testosterone and Follicle stimulating hormone (FSH) were measured
using ELISA kits obtained from Monobind Inc. Lake forest, CA, U.S.A and the
procedure was done as described by Adeleke et al. (2017).
Reverse Transcriptase Polymerase Chain Reaction Analysis
RNA was isolated from testis using the TRIzol Reagent (ThermoFisher Scientific).
Purified DNA-free RNA was converted to cDNA immediately using
ProtoScript® First Strand cDNA Synthesis Kit (NEB). Total cDNA
(5µL, 10ng) was subjected to PCR amplification in a 50µL reaction
mixture containing 10-µL PCR buffer (10mM Tris-HCl, pH 8.4, 50mM KCl/1.5
mM MgCl2), 2.5µL (10 mM) each, deoxynucleotide triphosphate, 5µL
each of forward and reverse (10mM) primers. Amplification conditions were:
Pre-denaturation at 94ºC for 5 min, Denaturation at 94ºC for 30 sec, Annealing
at 58ºC for 30 sec and Extension at 72ºC for 30 sec then 5 min at 72ºC by 30
cycles. The amplicons generated during the PCR step were resolved on 5% Agarose
gel. In-gel expression bands were captured using the iPhone-5c camera (Noir
effect). Gel image post-processing was done on the Keynote platform in a MacBook
Pro iOS computer. The densitometry analysis was carried out using the Image-J
software (2.3.0 V, Mac version), and finally the bar chart showing the relative
expression of target genes was done on the Graphpad Prism platform (version
8.03, for Mac iOS).
Statistical Analysis
We used the GraphPad Prism version 8.03 for all the statistical analyses. All the
data was expressed as Mean±SEM while differences among groups were
analyzed by one-way ANOVA. Tukey’s test was used to adjust for multiple
comparisons. p-value <0.05 was considered to be
statistically significant.
RESULTS
CREM, PROTAMINE I and II gene expression
Results depicted in Figure 1 revealed CREM
gene expression mean values after the administration of Chlorpromazine,
Reserpine, Rauwolfia vomitoria and Co-administration of
Reserpine, Ascorbate and Zinc. There was a significantly high upregulation of
the CREM gene with the administration of Rauwolfia vomitoria and co-administration of Reserpine, Ascorbate and Zinc (Groups F, G and H), when
compared with the control group A rats. Moreover, there was a significant
downregulation of the CREM gene expression in the 2.5mg/kg reserpine treated
group when compared with the control group A rats.
Figure 1.
Comparison in CREM gene expression among the groups after the
administration of Chlorpromazine, Reserpine, Rauwolfia
vomitoria and Co-administration of Reserpine, Ascorbate
and Zinc. a=Comparison with Control Group A; b=Comparison with Group
H and c=Comparison with group I. **p<0.01;
***p<0.001 (n=5).
Comparison of the CREM gene expression in the Chlorpromazine, Reserpine and
Co-administration of Reserpine, Ascorbate and Zinc treated groups showed high
significant down-regulation of CREM gene expression in Chlorpromazine and
Reserpine treated groups (Groups B, C, D, and E) when compared with
co-administration of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5 mg/kg Zinc
treated group H. Furthermore, there was a significant downregulation of the CREM
in the 2.5 and 5mg/kg-reserpine treated groups D and E, while there was
significant up-regulation in the 150mg/kg Rauwolfia vomitoria and co-administration of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5mg/kg Zinc
treated group H when compared with the co-administration of 5mg/kg Reserpine,
200mg/kg Ascorbate and 10 g/kg Zinc treated group I.
Figure 2 shows the Protamine 1 gene
expression mean values among experimental groups, with high significant
upregulation of the PRM-1 gene with 10 mg/kg Chlorpromazine, 5 mg/kg Reserpine,
300 mg/kg Rauwolfia vomitoria and co-administration of
Reserpine, Ascorbate and Zinc treated groups when compared with the control
group A rats. In Addition, there was a slightly significant up-regulation in the
150mg/kg Rauwolfia vomitoria treated group when compared with
the control group A.
Figure 2.
Comparison of the PRM-1 gene expression among the groups after
the administration of Chlorpromazine, Reserpine, Rauwolfia
vomitoria and Co-administration of Reserpine, Ascorbate
and Zinc. a=Comparison with Control Group A; b=Comparison with Group
H and c=Comparison with group I.*p<0.05;
**p<0.01; ***p<0.001
(n=5).
There was a highly significant downregulation of the PRM-1 gene expression with
5, 10mg/kg Chlorpromazine, 2.5mg/kg Reserpine and 150mg/kg Rauwolfia
vomitoria treated groups when compared with the co-administration
of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5 mg/kg Zinc treated group H.
Moreover, a slightly significant downregulation of the PRM-1 gene in the
300mg/kg Rauwolfia vomitoria treated groups when compared with
co-administration of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5mg/kg Zinc
treated group H.
Furthermore, Chlorpromazine, Reserpine and Rauwolfia vomitoria treated groups B, C, D, E, F and G had a highly significant downregulation of
the PRM-1 gene expression when compared with co-administration of 5mg/kg
Reserpine, 200mg/kg Ascorbate and 10mg/kg Zinc treated group I.
Figure 3 shows the mean values of
Protamine-2 gene expression, revealing a slightly significant downregulation of
the PRM-2 gene expression in the 2.5mg/kg Reserpine treated group when compared
with the control group A and co-administration of 2.5mg/kg Reserpine, 100mg/kg
Ascorbate and 5 mg/kg Zinc treated group H. In addition to this, a highly
significant downregulation of the PRM-2 gene expression was found in the 5mg/kg
Reserpine treated group when compared with the control group A, and treated
group H with co-administration of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and
5mg/kg Zinc.
Figure 3.
Showed comparison in PRM-2 gene expression among the groups after
the administration of Chlorpromazine, Reserpine, Rauwolfia
vomitoria and Co-administration of Reserpine, Ascorbate
and Zinc. a=Comparison with Control Group A; b=Comparison with Group
H and c=Comparison with group I.*p<0.05;
**p<0.01; ***p<0.001
(n=5).
Comparison of PRM-2 gene expression between the co-administration of 5 mg/kg Reserpine, 200mg/kg Ascorbate and 10mg/kg Zinc treated group I and other treated groups showed significant downregulation of the PRM-2 gene expression in 5, 10mg/kg Chlorpromazine and 300mg/kg Rauwolfia vomitoria treated groups. Furthermore, a highly significant down-regulation of the PRM-2 gene expression were seen in the 2.5, 5mg/kg Reserpine and 150mg/kg Rauwolfia vomitoria treated groups.
Hormonal analysis for Follicle Stimulating Hormone and Testosterone
Result from Figure 4 showed mean values for
serum follicle stimulating hormone concentration after administration of
Chlorpromazine, Reserpine, Rauwolfia vomitoria and
Co-administration of Reserpine, Ascorbate and Zinc. Slight significant decrease
in serum FSH concentration was observed in 10mg/kg Chlorpromazine treated group
while moderate significant decrease was observed in 5mg/kg of Reserpine treated
group when compared with the control group A.
Figure 4.
Showed comparison in Serum FSH Concentration among the groups
after the administration of Chlorpromazine, Reserpine, Rauwolfia vomitoria and Co-administration of
Reserpine, Ascorbate and Zinc. a=Comparison with Control Group A;
b=Comparison with Group H and c=Comparison with group
I.*p<0.05; **p<0.01;
***p<0.001 (n=5).
Serum FSH concentration was significantly decreased in 5mg/kg and 2.5mg/kg
Chlorpromazine and Reserpine respectively when compared with co-administration
of 2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5mg/kg Zinc treated group H.
Furthermore, high significant decrease in serum FSH were noticed in 10mg/kg
Chlorpromazine and 5 mg/kg Reserpine when compared with co-administration of
2.5mg/kg Reserpine, 100mg/kg Ascorbate and 5mg/kg Zinc treated group H.
Comparison amongco-administration of 5mg/kg Reserpine, 200mg/kg Ascorbate and
10mg/kg Zinc treated group I with synthetic antipsychotic drugs groups B (5mg/kg
Chlorpromazine), C (10mg/kg Chlorpromazine), D (2.5mg/kg Reserpine) and E
(5mg/kg Reserpine) showed high significant decrease in their serum FSH
concentration when compared with group I.
Figure 5 graph revealed mean values of
serum testosterone concentration among the groups after the administration of
Chlorpromazine, Reserpine, Rauwolfia vomitoria and
Co-administration of Reserpine, Ascorbate and Zinc. Serum testosterone were
significantly decreased in 10mg/kg Chlorpromazine and 5mg/kg Reserpine treated
groups while high significant increase was observed in 300mg/kg Rauwolfia vomitoria, co-administration of 2.5mg/kg
Reserpine, 100mg/kg Ascorbate and 5mg/kg Zinc and co-administration of 5mg/kg
Reserpine, 200mg/kg Ascorbate and 10mg/kg Zinc treated groups when compared with
control group A.
Figure 5.
Comparison in Serum Testosterone Concentration among the groups
after the administration of Chlorpromazine, Reserpine, Rauwolfia vomitoria and Co-administration of
Reserpine, Ascorbate and Zinc. a=Comparison with Control Group A;
b=Comparison with Group H and c=Comparison with group
I.*p<0.05; **p<0.01;
***p<0.001 (n=5).
Moreover, comparison among co-administration of 2.5mg/kg Reserpine, 100mg/kg
Ascorbate and 5mg/kg Zinc treated group A and co-administration of 5 mg/kg
Reserpine, 200mg/kg Ascorbate and 10mg/kg Zinc treated group I with synthetic
antipsychotic drugs groups B (5mg/kg Chlorpromazine), C (10mg/kg
Chlorpromazine), D (2.5mg/kg Reserpine) and E (5mg/kg Reserpine) revealed high
significant decrease in serum LH concentration in all synthetic antipsychotic
drugs treated groups.
Prolactin mean values as depicted in Figure
6 revealed high significant increase in serum prolactin concentration
in synthetic antipsychotic treated groups B (5mg/kg Chlorpromazine), C (10mg/kg
Chlorpromazine), D (2.5mg/kg Reserpine) and E (5mg/kg Reserpine) when compared
with the control group A, co-administration of 2.5mg/kg Reserpine, 100mg/kg
Ascorbate and 5 mg/kg Zinc and co-administration of 5mg/kg Reserpine, 200mg/kg
Ascorbate and 10mg/kg Zinc treated groups. More also, no significant difference
was observed among control group A, co-administration of 2.5mg/kg Reserpine,
100mg/kg Ascorbate and 5mg/kg Zinc and co-administration of 5mg/kg Reserpine,
200mg/kg Ascorbate and 10mg/kg Zinc treated groups.
Figure 6.
Comparison in Serum Prolactin Concentration among the groups
after the administration of Chlorpromazine, Reserpine, Rauwolfia vomitoria and Co-administration of
Reserpine, Ascorbate and Zinc. a=Comparison with Control Group A;
b=Comparison with Group H and c=Comparison with group
I.*p<0.05; **p<0.01;
***p<0.001 (n=5).
DISCUSSION
Spermatogenesis has been a complex procedure, involving mitotic, meiosis division and
differentiation of spermatogonial stem cells into mature spermatozoa. Phases of
spermatogenesis process are mitosis proliferation of spermatogonial stem cells to
produce spermatocytes, spermatocyte undergo meiosis division to form haploid round
spermatids, while the final stage called spermiogenesis involve conversion of round
spermatids to mature elongated spermatids (He et al., 2009). Male infertility (abnormal
spermatogenesis) is a pressing issue now, thus promoting an urgent need to solve
this fertility deficit. This research study underscores the efficacy of some target
antipsychotic compounds in driving the expression of some putative genes involved in
normal spermatogenesis.
cAMP responsive element modulator (CREM) is a member of the basic domain-leucine
zipper class of transcription factor, which binds as homo and heterodimers to a
regulatory palindromic DNA sequence, the cAMP response element (CRE). CRE is
localized in the promoter regions of the cAMP responsive genes. The absence of
CREM-dependent Transcription in post-meiotic germ cells results in an arrest of
spermatid differentiation and apoptosis (Hogeveen
& Sassone-Coris, 2006). Several spermatid-specific genes are known to
contain a cAMP-responsive element (CRE) serving as a binding site for the
transcription factor cAMP-responsive element modulator (CREM) (Sassone-Corsi, 1995). CREM is essential for spermatogenesis,
since it has been reported that male mice lacking a functional CREM gene are sterile
due to round spermatid maturation arrest (Blendy et al., 1996; Nantel et al., 1996). Infertile men exhibiting round
spermatid maturation arrest reveal a substantial reduction or a complete lack at the
level of both CREM protein (Weinbauer et
al., 1998) and CREM mRNA (Steger, 1999). Due to alternative transcriptional start sites,
alternative transcript splicing, and alternative translational start sites, the CREM
gene gives rise to functionally different proteins with either activating or
repressing potential on target gene expression (Daniel et al., 2000; Behr & Weinbauer, 2001; Gellersen et al., 2002). Weinbauer et al. (1998) reported that the alterations
in CREM expression, which interfere with the spermatid maturation within a number of
cases in idiopathic male infertility.
Protamines are post-meiotic nuclear proteins, which are rich in Arginine. At late
haploid phase of spermatogenesis, protamine substitute histones and help in
stabilizing sperm DNA and sperm head condensation. During spermatogenesis, haploid
spermatids will experience a transformation in its chromatin composition and
compactness (Steger, 1999), whereas the
deoxynucleic acid (DNA)-histone bond in the round spermatid will be substituted with
transition proteins; while the transition protein in elongated spermatids will be
substituted with protamine. Thus, changing from histone to protamine stimulates
spermatozoa chromatin condensation (Steger et al., 2001). Protamine has been experimentally
documented to be essential in male's fertility. Insufficient PRM-1 and PRM-2
concentrations have been implicated in subfertile or severe infertile condition
(Oliva, 2006).
From this section we investigate the gene expression pattern of CREM, PRM I and II
among the groups after the administration of Chlorpromazine, Reserpine, Rauwolfia vomitoria and Co-administration of Reserpine, Zinc
and Ascorbate. There was significant downregulation of the CREM and PRM II gene
expression in the Chlorpromazine and Reserpine treated groups when compared with the
control group. This result is in line with studies from Raji et al. (2005) and Khazan et al. (1960), who reported
anti-fertility effects of Chlorpromazine and Reserpine respectively. Moreover, there
was a slightly significant upregulation of the CREM, PRM I and II genes in groups F
and G treated with traditionally used antipsychotic drugs (Rauwolfia
vomitoria leaves extract). Furthermore, groups H and I treated with the
co-administration of Reserpine, Zinc and Ascorbate showed highly significant
up-regulation of the CREM, PRM I and II genes expression. The CREM gene expression
upregulation in groups F, G, H and I might be the result of antioxidant compounds
(Zinc and Ascorbate) being part of the drugs constituent administered to these
groups. This result is in agreement with those from Ahmadi et al. (2016) and Shabanian et al. (2017), who reported ascorbic
acid to be an important antioxidant that helps prevent sperm defects and boosts
sperm motility. Likewise, Ahmadi et
al. (2016) and Fallah et al. (2018) reported efficacy of Zinc supplements
in improving sperm count, motility, form, function, quality and fertilizing
capacity.
There are reports that CREM alteration or deficiency affect protamine expression;
thereby leading to infertility because of disorder in the round spermatid maturation
(Blendy et al., 1996).
The results from the gene-regulatory based approach in the current study correlate
with the reported connection with the putative genes that enhance male normal
fertility by Blendy et al. (1996).
Serum FSH and Testosterone concentrations were significantly decreased in the high
dose synthetic antipsychotic treated groups C and E (Chlorpromazine and Reserpine
respectively); while there was no significant difference in the Rauwolfia
vomitoria treated groups F and G when compared with the control group
A. Moreover, there was a highly significant increase in Serum FSH and Testosterone
concentrations with the co-administration of Reserpine, Zinc and Ascorbate. FSH and
testosterone concentrations found in these groups were in accordance with the
reports from Zamani et al. (2015), who reported a significant increase in serum prolactin and a
significant decrease in testosterone and luteinizing Hormone after Chlorpromazine
treatment in rats. Furthermore, Serum Prolactin concentrations in high dose
Chlorpromazine and Reserpine treated groups (C and E) were highly significantly
increased, and this report is in line with Ben-Jonathan & Hnasko (2001), who reported that dopamine depletion
reaching the lactotroph cells resulted in hyperprolactinemia, which always has
negative feedback on hypothalamic GnRH secretion.
Reproductive toxicity induced by Chlorpromazine and Reserpine from the results above
might come from dopaminergic fibers projecting from A13 and A14 of the ventral
tegmental area to the Paraventricular and medial preoptic nuclei of the
hypothalamus, respectively (Weiner & Molinoff,
1989). Thus, inhibition or depletion in dopamine concentrations in these
hypothalamic areas, because of antipsychotic drugs, may alter pulsatile production
of GnRH in the media preoptic area, with its resultant effects felt on the FSH and
LH production in the adenohypophysis. FSH has been reported to be a key player in
the transcription process of the CREM gene, absence of CREM-dependent transcription
in post-meiotic germ cells results in an arrest of spermatid differentiation and
apoptosis (Sassone-Corsi, 1998). FSH
predominantly regulates the CREM mRNA level. FSH binds with the G-protein
alpha-s-coupled receptors, such as Follicle stimulating hormone receptor (FSHR) and
activates Adenylate cyclase, Protein kinase A and cAMP-dependent PKA.
The CREM gene consists of CRE regions in the promoters, and its expression is
regulated by another cAMP-responsive element binding to protein 1 (CREB1); CREM
expression is alternatively regulated by an autoregulation pathway (Monaco et al., 1995; Hogeveen & Sassone-Coris, 2006). A germ
cell-specific transcriptional co-activator with four and a half LIM domains, with 5
(ACT) interactions, controls CREM activity. ACT capacity to control CREM activity is
regulated by a germ cell-specific kinesin, the Kinesin family member 17 (KIF17),
which regulates the ACT subcellular localization. KIF17 colocalizes with ACT in
haploid spermatids and mediates the ACT transport from the nucleus to the cytoplasm
at specific stages of the spermatid maturation. KIF17 movement is modulated by PKA
phosphorylation (c-AMP-dependent). The ability of KIF17 to shuttle between the
nuclear and the cytoplasmic compartments and to transport ACT are dependent on
neither its motor domain nor on microtubules (Kotaja et al., 2005; Hogeveen & Sassone-Coris, 2006). Thus, CREM activation by ACT is
responsible for the transcription of many key genes in postmeiotic germ cells, such
as PRM I and PRM II, which are both responsible for DNA condensation and spermatid
quality during spermatogenesis.
CONCLUSION
This present study has elucidated the reproductive toxicity induced by synthetic
antipsychotic drugs (Chlorpromazine and Reserpine) and it also gives credence to the
activity of compounds present in the traditionally used antipsychotic herb
(Rauwolfia vomitoria) via crude extract administered, and the
concurrent administration of isolated phytochemicals (Reserpine, Zinc and Ascorbate)
to improve the menace of infertility associated with antipsychotic drugs by
upregulating the CREM, PRM I and II signaling pathways in an animal model. Thus, we
concluded that healthy and quality spermatozoa are essential for fertilization.
Thus, alteration in the signaling pathways of the genes responsible for
spermatogenesis may result in the production of low quality spermatozoa.
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