Iprodione and Chlorpyrifos, Alone and in a Mixture, Impaired Male Fertility and Sexual Behavior in Adult Rats via Suppression of Steroidogenic Genes and SIRT1/TERT/PGC-1α Pathway


 There is cumulative evidence that the iprodione (IPR) fungicide and the chlorpyrifos (CPF) insecticide are endocrine disruptors that can evoke reproductive toxicity. Yet, the underlying mechanisms are still unclear. Besides, the outcomes of their co-exposure to male sexual behavior and male fertility are still unknown. The effects of IPR (200 mg/kg b.wt) and CPF (7.45 mg/kg b.wt) single or mutual exposure for 65 days on sexual behavior, sex hormones, testicular enzymes, testis, and accessory sex gland histomorphometric measurements, apoptosis, and oxidative stress biomarkers were investigated. In addition, expression of nuclear receptor subfamily group A (NR5A1), 17β-hydroxysteroid dehydrogenase (HSD17B3), silent information regulator type-1 (SIRT1), telomerase reverse transcriptase (TERT) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) genes have been assessed. Our results revealed that the individual or concurrent IPR and CPF exposure significantly disturb the sexual behavior, semen characteristics, testicular enzymes, and male hormones level. Oxidative stress caused by IPR and CPF activates apoptosis by inducing Caspase 3 and reducing Bcl-2. Downregulation of HSD17B3, NR5A1, and SIRT1/TERT/PGC-1α pathway was evident. Of note, most of these disturbances were exaggerated in rats co-exposed to IPR and CPF compared to IPR or CPF alone. Conclusively, our findings verified that IPR and CPF possibly damage the male reproductive system and concurrent exposure should be avoided.


Introduction
Every year, almost 3 billion kg of pesticides, with a budget of about 40 billion USD, is used globally (Sharma et al. 2020). Consequently, many pesticides known as endocrine disruptors are continuously and simultaneously introduced to human and animals, which affect fertility and sexual differentiation (Ma et al. 2019). People are most frequently exposed to a mixture of pesticides by air, water, food and contaminated milk (Mehrpour et al. 2014, Rizzati et al. 2016. Likewise, insecticides are frequently mixed with fungicides in practice for simultaneous use in agricultural elds (Visalakshmi et al. 2016).
Organophosphorous insecticides are among the most worldwide-used agrochemicals (Kumar et al. 2018). In recent decades, Chlorpyrifos (CPF) is one of the most extensively used insecticides in both agricultural and domestic elds worldwide (Perez-Fernandez et al. 2020).. Exposure to CPF induced several harmful impacts like endocrine disruption (Watts 2012), hepatotoxicity (Xu et al. 2017), neurotoxicity (Burke et al. 2017), nephrotoxicity (Xu et al. 2018), and developmental toxicity (Laporte et al. 2018). Also, CPF has been reported to impair male reproduction ). The potential underlying mechanisms of CPF induced reprotoxic effects remain unclear .
Fungicides are considered a critical component in fruits and vegetable production (Li et al. 2009). Iprodione (IPR) is one of the dicarboxamide fungicides, widely used for fungal control in many crops (Grabke et al. 2014). The available data about IPR toxicity are limited and do not determine risk assessments (Chaufan et al. 2019). IPR showed anti-androgenic activity by reducing testosterone levels and delay in pubertal development in the male rat (Blystone et al. 2007). Also, IPR modi es estrogens and androgens synthesis (Pisani et al. 2016). Nevertheless, studies so far have yet to recognize the underlying mechanisms of IPR induced reprotoxic effects.
In Egypt, IPR is widely used to control fungal diseases. Various studies have been performed on IPR residues in some fruits and vegetables, such as strawberries, apples, onions, peas, green beans, and chilli peppers ( CPF is frequently detected in vegetables, cereals, and fruits (Angioni et al. 2011, Li et al. 2015. IPR and CPF have recently been detected by screening pesticide residues in cucumber crops in Egypt (Ahmed et al. 2019).
Various environmental pollutants have been reported to affect steroidogenic gene activities (Zimmer et al. 2011). Nuclear receptor subfamily group A (NR5A1) is a primary steroidogenic gene which plays a dominant role in the gonads development (Röpke et al. 2013). Also, 17β-hydroxysteroid dehydrogenase (HSD17B3) gene is a crucial regulator of steroidogenesis responsible for reducing active steroid hormones (Jana et al. 2006 (Lin &Elledge 2003). Also, impaired TERT expression has been implicated in spermatogenic failure (Weikert et al. 2006). The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) plays a main role in the regulation of mitochondrial biogenesis and function.
Inactivated PGC-1α is converted by SIRT1 to the active form. TERT, indeed a vital component of telomerases, can also regulate PGC-1α (Sahin et al. 2011).
Therefore, this study investigates two main issues. First, assess the outcome of the single or co-exposure of IPR and CPF on sexual behavior, sexual hormones, testicular enzymes, testis, accessory gland architecture, oxidative stress, and apoptosis biomarkers. Second, determine whether the IPR and/or CPF induced reprotoxic effect could be related to the modulation of NR5A1, HSD17B3 and SIRT1/TERT/ PGC-1α pathway in the testicular tissue. mg/kg b. wt. CPF. All treatments were given orally using gastric gavage for 65 successive days. All rats were weighed every week, the weight is recorded, and the dose volumes were determined accordingly.

Materials And
Throughout the experiment, the rats were closely monitored for discomfort, pain, mucosal membrane color, morbidity, and mortality.

Assessment of sexual behavior
Male rats were tested three times for male sexual activity to de ne those showing ejaculation at least two times. Behavioral tests were carried out in faint red lights. Male sexual behavior was evaluated by keeping the male in a Plexiglas arena ve minutes before presenting a receptive female. Female rats were got into sexual receptivity via subcutaneous injection of 10 µg estradiol benzoate dissolved in 100 µl oil before sexual trial by 44 h. Then, 1 mg of progesterone / 200-µl oil was subcutaneously injected for females four h before testing. The test continued for 30 min after the female presentation, and the many indicators were documented. Latencies to the rst mount, the rst intromission, and the rst ejaculation were recorded. The number of mounts (mounts with pelvic thrusting) and intromissions (mounts with pelvic thrusting and penile insertion) of the rst copulatory succession were counted. Furthermore, the frequency of ejaculation (ejaculation number throughout 30 min of recording) and post-ejaculatory interval (time between ejaculation and following intromission) were documented (Hull et al. 2006). The observations were recorded using a video camera connected to a monitor in a neighboring room.

Semen evaluation
Evaluation of semen was carried out as previously reported by (Arisha &Moustafa 2019). Brie y, The cauda epididymis was extracted from one testis and macerated in a petri dish containing 2 ml of prewarmed physiological saline solution at 37 o C (Hafez 1970). To determine the individual motility of the spermatozoa, one drop of the resultant solution was directly examined under a light microscope on prewarmed glass slides with a magni cation of 400×. The total percentage of motile sperm was measured in numerous microscopic elds. Total sperm count was estimated following dilution with physiological saline at 1:4 with addition of 5 drop of formalin (40%) solution. The total sperm count was calculated as the sum of spermatozoa counted in ve secondary square × 2500 × dilution rate. Sperm abnormalities were detected in eosin-nigrosine stained slides and counted per 100 sperm (Filler 1993, Seed et al. 1996 2.5. Blood and tissue sampling After 65 days, the rats were weighed and fasted overnight. From the medial canthus of the eye, blood samples were collected into a plain tube, permitted to coagulate for 30 minutes, and centrifuged at 3000 rpm for 10 minutes to separate serum for further biochemical evaluations. Following this, the rats were euthanized by cervical dislocation. Then the testes were directly excised and weighed. Samples from the testis were divided into three portions. The rst part homogenized in phosphate-buffered saline, centrifuged for 15 minutes at 3000×g, then the collected supernatant was kept frozen for oxidative stress markers analysis. The second portion was kept for histopathological screening after xing in phosphatebuffered formalin10%. The third portion was kept for gene expression investigations in liquid nitrogen at -80°C.

Measurement of oxidative stress and apoptosis biomarkers in testis homogenates
Total antioxidant capacity (TAC) was evaluated using the colorimetric technique, as described by Koracevic et al. (2001). Glutathione peroxidase (GPx) was assessed, according to Pascual et al. (1992). Reduced glutathione (GSH) level was determined according to the method described by Beutler et al. (1963). Malondialdehyde (MDA) was measured, according to Esterbauer et al. (1982). B-cell CLL/lymphoma 2 (Bcl2) and Caspase 3 were quantitated using ELISA Kit (MyBioSource, San Diego, California, USA, Catalog No: MBS704330 and MBS700575, respectively) in line with the manufacturer's directions.

Histopathological examination and histomorphometric analysis
All tissue samples, including testes, cauda epididymis, prostate and seminal vesicles were kept in formalin solution 10% and subsequently dehydrated, cleared in xylene, embedded, and blocked in para n using an automatic tissue processor. Then, 5µm thickness sections were cut by a rotary The mean epithelial height and bro-muscular layer thickness (FMT) were measured in the seminal vesicles sections from each rat (Justulin et al. 2006

Effects on sexual behavior
Data on sexual behavior evaluation are displayed in Table 2. Exposure to IPR alone induced a signi cant delay in the mounting latency and inter intromission interval compared with the control group. At the same time, mount and ejaculation latencies, ejaculation frequency together with post-ejaculatory inter intromission intervals, were signi cantly increased in rats exposed to CPF alone compared to the control. Notably, the co-exposure group showed a signi cant increase in mount, intromission, and ejaculation latencies, ejaculation frequency, and post-ejaculatory inter intromission intervals in relation to the control group. The intromission ratio was signi cantly reduced in all the treated groups compared to the control one. Nevertheless, both mount and intromission frequencies were not signi cantly changed among the different treated groups.  3.2. Effects on body weight, testis weight, and sperm parameters As documented in Table 3, oral dosing of IPR, CPF, and their combination for 65 days triggered signi cant decrease in the body weight change, and testis weight compared to the control group. Moreover, the gonadosomatic index was signi cantly reduced in IPR and IPR + CPF exposed rats. The sperm motility percentage and sperm count of IPR, CPF, and IPR + CPF groups showed a signi cant reduction compared with the control group. However, sperm abnormalities% increased signi cantly increased in rats individually or simultaneously exposed to IPR and CPF compared to control rats. Notably, the IPR + CPF group showed a signi cant difference in sperm abnormalities % compared with IPR or CPF exposed groups. These morphological alterations comprise a detached tail, looped tail, abnormal hookless head, coiled tail, curved tail, detached head, bent neck, bent neck with a hookless head, as demonstrated in Fig. 1.

Effects on serum hormonal pro le and testicular enzymes
The obtained data in Table 4 showed that serum Testosterone, LH, FSH and E2 were signi cantly reduced in rats individually or concurrently exposed to IPR and CPF compared with control ones. Furthermore, serum ACP and SDH levels in all the treated rats were signi cantly decreased compared with control rats.

Effects on oxidative stress and apoptosis markers
The administration of IPR, CPF, and IPR + CPF lead to a signi cant reduction in the TAC, GSH, and GPx levels but a substantial increase in the MDA level compared to the control values. Regarding the apoptotic markers, the obtained data demonstrated a signi cant increase in Caspase 3 levels; while Bcl-2 was signi cantly decreased especially in IPR + CPF group.
3.5. Histopathological ndings 3.5.1. Testicular tissue The testicular tissue of control rats revealed well-developed seminiferous tubules with active spermatogenesis at different stages of germinal cell differentiation and maturation. There were prominent interstitial Leydig cells. On the other side, testicular tissue sections of IPR and CPFadministered rats showed testicular alterations with spermatogenesis interruption. These alterations were more prominent in the CPF-administered group, which were represented by degenerative changes of spermatogenic and Sertoli cells accompanied by lumen contraction and thickened and corrugated basement membrane. There were desquamated germ cells with few sperm cells and little debris of spermatids in their lumen. Wide spaces separated between the degenerated germinal epithelium and the basement membrane inside the seminiferous tubules appeared. Besides, the co-exposed group showed severe tubular damages as degenerative changes and desquamation of their germinal epithelium associated with no or few spermatogenic cells in the atrophied tubules, leading to complete cessation of spermatogenesis.
Moreover, all treated groups showed vacuolar degenerated Leydig cells associated with leucocytic in ltrations in the interstitial tissues in different degrees. There was apparent interstitial blood vessel dilatation and congestion. General interstitial and sub-scapular edema were noticed that leading to wide vacuolated intertubular spaces. The testicular parenchyma was covered by a thick testicular capsule that contained congested blood vessels ( Fig. 2A-G).

Epididymis tissues
The examined sections of the control group showed normal tubular structure lled with a dense aggregation of sperms by light microscopy. On the other side, examining all treated rats' cauda epididymis revealed that tubular irregularity and lined with at epithelial cells. There was vacuole formation with disrupted stereocilia. Epithelial in ammation and necrosis in the epididymis were noted. There were marked pathological changes especially rats treated with CPF or with IPR. These changes included degeneration and cytoplasmic vacuolation with desquamated stereocilia of the principal cells.
Hypertrophy of the clear cells with a degenerated nucleus and increased vesicles were showed. Also, there was growth in halo cells with hypertrophy. In addition, a broken basement membrane with destructed tubules appeared. Increased cell debris and abnormal round germ cells associated with reduction or absence of spermatozoa density appeared in the epididymis lumen. The hypertrophied intertubular connective tissue had congested blood vessels, lymphocytic in ltrations, and edema. Moreover, thickened smooth muscle layers between the tubules were noted. The capsule was showed thickness accompanied by blood vessel congestion and subcapsular edema (Fig. 3A-G).

Prostate gland tissues
The prostate glands of the control group revealed no histopathological alterations. They appeared with normal acini enclosed in an outer capsule and lled with pink secretion. The different treated materials induced several histopathological changes associated with irregular acinar shape and size. The glandular tissues showed a marked degree of epithelial hyperplasia in most of the acini accompanied with papillary projections, and other alveoli were enlarged with abnormal attened epithelium. This hyperplasia was more prominent in both groups that treated with IPR or CPF alone. On the other side, there was marked epithelial hyperplasia and papillary projections accompanied by acinar shrinkage and narrow lumen in IPR and CPF-treated rats. Moreover, there were epithelial degeneration and necrosis, as well as luminal cellular debris. Thick bro-muscular stroma accompanied by vacuolations surrounded the acini in most of the examined glands. It was remarkable that interstitial spaces showed interstitial edema, blood vessels congestion, and vasculitis. There were hemorrhages and monocellular in ammatory cells in ltration inside the acinar lumen and around the acini. There were marked edema, hemorrhages and congested blood vessels in the prostatic capsule (Fig. 4A-G).

Seminal vesicle tissues
The seminal vesicles of the control group appeared normal. They showed epithelial folds that lined with columnar epithelium and basal cells. A bro-muscular layer followed this layer, and the gland was enclosed with a capsule. A secretion was visible in the gland lumen. While treated groups with IPR and CPF showed gland hyperplasia with papillary structures compared to those of control rats. Vacuolar degeneration and detached epithelium were noticed. A remarkable increase in gland secretions in the dilated lumen was also evident. On the other side, the seminal vesicles of IPR and CPF rats revealed that glandular atrophy with destructed folds and scanty uid in their lumen. Also, the bro-muscular layer of all treated groups showed edema with in ammatory cell in ltrations, mainly lymphocytes, and macrophages. There was blood vessel congestion accompanied by vasculitis. Vacuolar degeneration of this layer was prominent. Marked subcapsular edema was observed (Fig. 5A-F).
3.6. Morphometric analysis of testis and accessory glands 3.6.1. Morphometric analysis of testis As shown in Table 5, seminiferous tubules diameter was signi cantly decreased in the treated groups, especially in CPF group. It was observed that the testicular GEH and TLD values were signi cantly decreased in all treated groups. While testicular capsule thickness was highly increased in all treated groups compared to the control. The epididymis tubular diameter and length was signi cantly increased in all the treated groups. The EEH was signi cantly increased in CPF group while decreased in IPR and combination groups. Prostate epithelium height was slightly increased in IPR group and CPF group while decreased in the combination group. SMT of prostate was signi cantly increased in all treated groups. Seminal vesicle epithelial height and FMT was increased in all treated groups especially in the combination group (Table 5).

Gene expression ndings
As shown in Fig. 6, a signi cant down-regulation of NR5A1 and HSD17B3 mRNA levels in all treated groups and was obvious in IPR + CPF co-exposed group. All treated groups showed a signi cant downregulation of the SIRT1, TERT and PGC1α mRNA levels than the control.

Discussion
In our study, the IPR and CPF oral dosing induced a signi cant reduction in the body weight gain and testis weight, particularly in the group exposed to a combination of IPR and CPF. Oxidative stress may play a role in reducing body weight (Mossa et al. 2015). Reproductive organ weights are sensitive measures of chemical-induced reproductive toxicity (Zidan 2009). The reduction of testicular weights may be due to reduced tubular size, as con rmed by the testis' histopathological ndings. Also, inhibition of steroid biosynthesis of Leydig cells and arrest of spermatogenesis may be a possible cause of such reduction.
Herein, in rats exposed to IPR, a reduction in serum testosterone may be linked to the IPR imidazole-like ring. Many chemical compounds in the imidazole class inhibit steroidogenesis via one or more cytochrome P450s (Blystone et al. 2007). Furthermore, Rone et al. (2009) reported that IPR prevention of the movement of cholesterol from the external to the inner mitochondrial membrane might adversely affect the production of the sexual steroids. Besides, the sexual hormonal imbalance observed in CPF exposed rats may be owed to the direct degenerative effect on Leydig cells and/or indirectly via its nicotine-like suppressive activity on LH release from the pituitary gland as one of the acetylcholine receptors agonists (Peiris &Dhanushka 2017, Rato et al. 2015). Testicular marker enzymes, namely ACP and SDH, are essential to healthy germ cell growth and are closely associated with spermatogenesis (Adedara et al. 2018a). The substantial decrease in testicular function marker enzymes in rats exposed to IPR and CPF suggests a reduction in the transport of testicular nutrients, energy metabolism, and division into sperm cells (Adedara et al. 2018b) Concerning semen evaluation, the signi cant inhibition of sperm motility in all the exposed groups can be due to low ATP levels (Bai &Shi 2002, Heikal et al. 2014. Also, LH is essential for the functional epididymal maturation and acquisition of progressive Ca 2+ motility through the absorption of speci c bdefensin from the main cells. (Lehrer &Lu 2012). The decrease in LH level could, therefore, lead to reduced sperm motility observed in this study. The reduction in sperm count of the treated rats in our study may be linked to the decrease in serum testosterone levels, resulting in the suppression of spermatogenesis (Sharma et al. 2014). In the testes, decreases in sperm counts can also be due to oxidative stress (Shittu et al. 2012). Sperm abnormalities are mainly hormone-dependent. So, the low testosterone level may be the critical factor producing sperm deformities (Guido et al. 2014).
Notably, herein, exposure to IPR and CPF alone or in a combination considerably disturb sexual behavior as revealed by deferred latencies to the rst mount, rst intromission, and rst ejaculation, in addition to diminished ejaculations number. In adult mammals, male sexual activity is modulated by TES (Robbins 1996); and it is controlled by the hypothalamic-pituitary-testicular axis (Ågmo 1997

Conclusion
In conclusion, our ndings demonstrated the detrimental impacts of the single or combined exposure to IPR and CPF on the male sexual behavior and male reproductive system function and architecture.
Besides, our ndings afford a distinctive and intriguing perspective that excessive ROS generation following IPR and CPF exposure could promote steroidogenic genes downregulation and suppress SIRT1/TERT/PGC1α pathway. Such ndings explain the target organelle and molecular mechanisms by which IPR and CPF contribute to animal and human impaired fertility. We thank all participants in this study.

Declarations
Data availability: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding: Not applicable
Compliance with ethical standards: Con icts of interests: The authors declare no con icts of interest.
Ethical approval: The authors of the current study con rm that all the experimental procedures were conducted in strict compliance with the ethical standards of the NIH guidelines on the care and use of laboratory animals to minimize the suffering of the experimental animals throughout the acclimatization, experimentation, and sampling. The ethical standards were approved by the Institutional Animal Care and