Effect of administration of green tea extract on regeneration of the testicular parenchyma of Wistar rats submitted to heat stress

Objective: This study evaluated the effects of green tea extract (GTE) on the recovery of testicular parenchyma of adult Wistar rats submitted to heat stress (HS). Methodology: Animals (n=84) were distributed and treated, according to the experimental group (G1: unstressed and untreated; G2: stressed and untreated; G3: unstressed and treated; G4: stressed and treated). Results: The overall mean of testicular weight, epithelial height and tubular diameter were lower (P<0.05) in G2 and G4, and day 60 presented a higher value (P<0.05) to the two first parameters. The gonadosomatic index and the volumes of the seminiferous epithelium were lower (P<0.05) in G2 and G4. The overall means G2 and G4, although with higher values (P<0.05) on day 60. The volume of lymphatic space was higher (P<0.05) in G2 and G4, being day 60 higher (P<0.05) than day 15. Greater preservation and recovery of seminiferous epithelium occurred in G4, compared to G2. Conclusion: Thus, the GTE administration is an effective way to improving tissue recovery after testicular damage, induced by short-term heat.


Introduction
Reproduction is essential for the perpetuation of species (Durairajanayagam et al., 2015;Carvalho et al., 2021). However, reproductive performance is influenced by multiple factors, with temperature being one of the most prominent elements (Boni, 2019;Schou et al., 2021), because its variations causes alterations in physiological parameters, how spermatogenesis functions, with consequent fertility impairment (Durairajanayagam et al., 2015). Heat stress is a problem experienced daily, especially in countries with a tropical climate, what has worsened with the climate change, marked by the global warming (Sinha et al., 2017;Schou et al., 2021). Testicular degeneration can result from heat stress and determine spermatogenic cell death, defects in the DNA, and altered gene expression, with impairment in function and consequent failures in spermatogenesis (Lue et al., 1999). Structural and functional damage to male gonads and gametes, caused by exposure to excessive heat, are largely associated with the oxidative stress (Garcia-Oliveros et al., 2020) and reduction in antioxidant capacity (Fraczek et al., 2022), once that hyperthermia causes disruption of the oxidative homeostasis (Hamilton et al., 2016;Cilio et al., 2022;Wang et al., 2022).
As opposed to heat-induced damage and oxidizing agents, antioxidants may retard or inhibit oxidative stress, preventing the onset or continuation of the deleterious effects of this condition (Abshenas et al., 2011;Cilio et al., 2022). They can be acquired through diet such as green tea (Camellia sinensis), rich in polyphenolic compounds, especially catechins (Farhan, 2022;Nain et al., 2022), a class of flavonoids with antioxidant, anti-inflammatory, and anti-carcinogenic properties (Zanchi et al., 2015;Farhan, 2022), as well as inhibitor of enzymes, as those involved in the apoptotic cascade (Bagherpour et al., 2019). Thus, the objective of this study was to evaluate the effects of green tea extract on the regeneration of the testicular parenchyma of Wistar rats submitted to heat stress.

Methodology
The study was carried out at the Physiology and Pathology laboratories, of the Federal Rural University of Pernambuco (UFRPE), after approbation by the Ethics Committee for Animal Experimentation (CEEUA/UFRPE 059/2015). A total of 84 adult Wistar rats (Rattus norvegicus, var. Albinus) from the bioterium of the Department of Animal Morphology and Physiology (UFRPE) were kept in an environment at 23 ± 1ºC, 50% humidity, and a 12-h light-dark cycle. Water and rodent feed were offered ad libitum.
At 120 days of age, the animals were divided through random probabilistic sampling into four experimental groups, each with 21 subjects (G1: not supplemented and not exposed to heat stress; G2: not supplemented and exposed to heat stress; G3: supplemented with green tea extract and not exposed to heat stress; G4: supplemented with green tea extract and exposed to heat stress). In turn, green tea extract (China-Hong Kong, via Pharma Nostra® handling establishment) was weighed and diluted daily in distilled water, so that each G3 and G4 animal received, every 24 hours, 100 mg/kg of extract in aqueous solution form, based on the amount of 50 mg/kg epigallocatechin gallate present in the extract used (Vieira et al., 2020).
On day 0 of the study, the animals in the G2 and G4 groups were anesthetized intraperitoneally with 80 mg/kg of 10% Ketamine Hydrochloride (Syntec) and 6 mg/kg of Xylazine Hydrochloride 2% (Syntec), placed in a support and their testicles immersed in a water bath at 43 °C for 15 min. Subsequent to recovery from anesthesia, administration by oral gavage of aqueous solution of green tea extract or distilled water was initiated, according to the experimental groups. The treatment continued for 60 days, during which the animals were weighed daily to correct the administered dose of the extract, and without sample loss (Vieira et al., 2020).
Subgroups of animals (n=07 per group, totaling 28) were euthanized, through intraperitoneal administration of the anesthetic Thiopental Sodium 0.5 g, 50 mg/kg (Tiopentax® Cristália), on days 15, 30, and 60 after testicular heat stress and submitted to intracardiac perfusion with NaCl physiological solution (0.9%) for vascular lavage. Subsequently, the animals were perfused with 4% glutaraldehyde fixative solution (Vetec) in sodium phosphate buffer (pH 7.2 and 0.01 M). The testicles were removed and sectioned into fragments up to 2 mm thick, which were re-fixed in the same infusion solution, and immersed in a phosphate buffer for 2 h, dehydrated in increasing series of alcoholic solutions (70-100%), and incorporated in plastic resin composed of glycol methacrylate (Leica®). Histological sections 4 μm thick were used for slides preparation, stained with hematoxylin and floxin, and then analyzed morphologically and morphometrically.
The tubular diameter, height, and epithelial area were measured with an eyepiece micrometer (100X), calibrated with a standard micrometer. Twenty round or rounded tubular profiles were measured using the ImageJ program, with the mean determined for each animal. The morphometric data of the testicles composition were obtained using point counting, through random systematic allocation of an integrated graticule with 441 points of intersection, on the testicular parenchyma, with an increase of 400X. Approximately 6600 points were counted for each rat.
The volume of each testicle component was established from the product between the volumetric density (%) and the net volume of the testicle (net testicle weight). The specific gravity of the testicular tissue was considered as 1.0, for subsequent morphometric calculations. In order to calculate an exact measure of the net volume of the testicle, 6.5% of the capsule was extracted from its weight (Russell & França, 1995). The total length of the seminiferous tubules (meters) was obtained through the division of the tubule volume by πR2 (França et al., 2000). In addition, from the weight of both testicles, it was possible to calculate the gonadosomatic index, which refers to the percentage of body mass allocated in gonads (Caldeira et al., 2010).
The data were analyzed according to ProcMixed for mixed models (SAS Int. Inc., Cary, NY), with collection days as repeated measurements over time (15, 30, and 60 days). First, the data were analyzed in relation to the presence of normality of the residues (Shapiro-Wilk). Logarithmic or square root transformations (x+1) were necessary when the normality premise was not met. As for the data with response in percentage, they were submitted to angular transformation of the arcsine of the square root of the percentage value, according to Sampaio (1998). The statistical model includes a fixed treatment effect (T), days (D), and interactions (TxD). The means contrast was performed by the Student-Newman-Keuls test at the 5% probability level. The number of animals established in this design made it possible to determine the degrees of freedom, with treatments = k -1; total = kr -1; residue = k (r-1), establishing, in this context, an adequate number of repetitions per treatment.

Results
The overall mean of testicular weight, seminiferous tubules diameter and height of the seminiferous epithelium (Table   1) were lower (P<0.05) in the G2 and G4 than in the G1 and G3. However, higher values were observed on day 60 (P<0.05) than on days 15 and 30 of the experiment to the first and last parameters. In addition, the gonadosomatic index (Table 1) on days 15, 30, and 60 was lower (P<0.05) in the G2 and G4, than in the G1 and G3, independent of the administration of green tea extract. With regard to volumetric characteristics of the seminiferous epithelium (  In the histopathological analysis, 15 days after the testicular heat stress (Figure 1), the G2 group presented testicular degeneration, with loss of the germinal epithelium and desquamated cells. On the other hand, in G4 animals presented seminiferous tubules with reduced germ cell population and increased intertubular space.

Discussion
No involvement from testicular heat stress and/or administration of green tea extract was observed on the body weight of male Wistar rats, a fact that corroborated with Hijazi et al. (2015). However, this diverges from literature reports on the lipolytic and weight-reducing effect of this compound, although it is a dose-dependent factor, influenced too by the experimental conditions employed (Chandra et al., 2011, Baláži et al., 2022. The body weight of the animals from the different experimental groups varied from 345.14 to 389.57 g, remaining within the standards for the species and sex (360 to 416 g), as described by Oliveira et al. (2015).
A reduction in testicle weights is an evident sign of their exposure to heat, whether acute or chronic (Yarmolenko et al., 2011;Yadav et al., 2017;Ngoula et al., 2020). According to Kanter et al. (2013) and Lue et al. (1999), a reduction in the testicular weight of rats can be observed one or two days after being submitted to 43 ºC for 30 or 15 min, respectively, without recovery until day 35 (Kanter et al., 2013). This parameter resumes values similar to those of the control group only after 56 days (Lue et al., 1999). These observations are similar to that of the present study, where at 60 days after the heat shock the testicular weights were higher than on days 15 and 30, without positive or negative interference of the green tea extract.
The gonadosomatic index was widely compromised by the heat, regardless of the administration of green tea. How this parameter refers to the percentage of body mass allocated to the testicles of the animal (Capucho et al. 2022), its reduction is justified by the lower testicular weight of the heat stressed animals. Moreover, testicular hyperthermia decreased the diameter of the seminiferous tubules, like observed by Kanter et al. (2013) and Thanh et al. (2020), but without green tea protective effect, in opposition to Bagherpour et al. (2019) reports. Nevertheless, the mean tubule diameter indicates the development of the seminiferous epithelium (Kanter et al., 2013), representing an important parameter.
Regarding the volumetric characteristics of the testicular parenchyma, the lower volumes of the seminiferous epithelium and seminiferous tubules in the stressed groups, in relation to the groups without heat stress, confirm the harmful role of heat for the gonads, and the protective effect of the treatment. It is reinforced by the fact of EGCG, present in large amounts in green tea (Nain et al., 2022), can prevent inflammatory, apoptotic and oxidant effects in testicles (Al-Maghrebi et al., 2012;Bagherpour et al., 2019), with protection of the seminiferous tubules (Ding et al., 2015;Bagherpour et al., 2019).
The lymphatic space presented greater volume in the stressed groups than in the control group, due to the smaller diameter and height of seminiferous tubules as, consequently, the interstitial space becomes larger. Based on the foregoing, it can be seen that damages in the seminiferous tubules culminate in damages to spermatogenesis, increasing the possibility of infertility (Kanter et al., 2013;Thanh et al., 2020).
Although recovery of the seminiferous epithelium is possible, it depends on the presence of viable spermatogonia and Sertoli cells, from which improvement in the germinal epithelium is verified around 60 days after the removal of the cause of the stress (Gabaldi & Wolf, 2002). Thus, the temperature and time of exposure to testicular heat stress are determinant to the recovery of gonadal functions, since they are limiting factors to the survival of spermatogonia and Sertoli cells (Durairajanayagam et al., 2015).
Through the qualitative histological analysis on day 15, it was possible to identify that lesions of the testicular parenchyma, including Sertoli cells, were less intense in the group that received the green tea extract (G4) than in the group only exposed to elevated testicular temperature (G2), which was repeated on day 30. This was even more striking on day 60, when, while the G2 demonstrated typical findings of testicular degeneration, the G4 presented marked recovery in the spermatogenic process.
In addition to the above exposure, the presence of all stages of the seminiferous epithelium cycle, as verified on day 60 for the G4, is indicative of complete restoration of spermatogenesis (Jannes et al., 1998). Thus, the findings demonstrate the protective effect of the catechins present in green tea, as previously reported, against the negative effects of testicular hyperthermia (Abshenas et al., 2011;Vieira et al, 2020), as well as the sensitivity of germ cells and Sertoli cells to the action of heat (Kanter et al., 2013;Thanh et al., 2020).
The results corroborate with previous reports that oral administration of green tea extract, after testicular thermal shock in mice (42ºC/20 min), prevented the depletion of the seminiferous tubules, although the mechanisms by which this extract provides benefits to spermatogenesis are not fully elucidated (Abshenas et al., 2011). Thus, they has been largely associated to the antioxidant, anti-apoptotic (Bagherpour et al., 2019), and anti-inflammatory actions (Al-Maghrebi et al., 2012) of the EGCG.
Therefore, research should be developed to prove the therapeutic activities and understand the mechanisms of the action of green tea, especially because this bioactive shows as an attractive alternative in the preservation of male fertility.

Conclusion
The administration of green tea extract is an effective way of improving tissue recovery after testicular damage induced by short-term heat stress in rat testicles, as it promotes full recovery of spermatogenesis in the second spermatogenic cycle.