Neonatal hypothyroidism does not increase Sertoli cell proliferation in iNOS−/− mice

in Reproduction
Correspondence should be addressed to L R França; Email: lrfranca@icb.ufmg.br

Sertoli cell (SC) proliferation in mice occurs until two weeks after birth and is mainly regulated by FSH and thyroid hormones. Previous studies have shown that transient neonatal hypothyroidism in laboratory rodents is able to extend SC mitotic activity, leading ultimately to higher testis size and daily sperm production (DSP) in adult animals. Moreover, we have shown that due to higher SC proliferation and lower germ cell apoptosis, iNOS deficiency in mice also results in higher testis size and DSP. Although the cell size was smaller, the Leydig cells (LCs) number per testis also significantly increased in iNOS−/− mice. Our aims in the present study were to investigate if the combination of neonatal hypothyroidism and iNOS deficiency promotes additive effects in SC number, testis size and DSP. Hypothyroidism was induced in wild-type (WT) and iNOS−/− mice using 6-propyl-2-thiouracil (PTU) through the mother’s drinking water from 0 to 20 days of age, and were sacrificed at adulthood. Our results showed that, in contrast to the WT mice in which testis size, DSP and SC numbers increased significantly by 20, 40 and 70% respectively, after PTU treatment, no additive effects were observed for these parameters in treated iNOS−/− mice, as well as for LC. No alterations were observed in spermatogenesis in any group evaluated. Although we still do not have an explanation for these intriguing findings, we are currently investigating whether thyroid hormones influence iNOS levels and/or counterbalance physiological effects of iNOS deficiency in testis function and spermatogenesis.

Abstract

Sertoli cell (SC) proliferation in mice occurs until two weeks after birth and is mainly regulated by FSH and thyroid hormones. Previous studies have shown that transient neonatal hypothyroidism in laboratory rodents is able to extend SC mitotic activity, leading ultimately to higher testis size and daily sperm production (DSP) in adult animals. Moreover, we have shown that due to higher SC proliferation and lower germ cell apoptosis, iNOS deficiency in mice also results in higher testis size and DSP. Although the cell size was smaller, the Leydig cells (LCs) number per testis also significantly increased in iNOS−/− mice. Our aims in the present study were to investigate if the combination of neonatal hypothyroidism and iNOS deficiency promotes additive effects in SC number, testis size and DSP. Hypothyroidism was induced in wild-type (WT) and iNOS−/− mice using 6-propyl-2-thiouracil (PTU) through the mother’s drinking water from 0 to 20 days of age, and were sacrificed at adulthood. Our results showed that, in contrast to the WT mice in which testis size, DSP and SC numbers increased significantly by 20, 40 and 70% respectively, after PTU treatment, no additive effects were observed for these parameters in treated iNOS−/− mice, as well as for LC. No alterations were observed in spermatogenesis in any group evaluated. Although we still do not have an explanation for these intriguing findings, we are currently investigating whether thyroid hormones influence iNOS levels and/or counterbalance physiological effects of iNOS deficiency in testis function and spermatogenesis.

Introduction

Sertoli cells (SCs) are the first somatic element to differentiate in the testis and have a pivotal importance in coordinating the development of this organ (DiNapoli & Capel 2008). This key testis somatic cell actively proliferates in foetal life, reaching its high mitotic activity just before birth (Orth 1982, 1993, McCoard et al. 2003, França et al. 2016), and continues to divide postnatally for about two weeks in mice and rats (Steinberger & Steinberger 1971, Orth 1982, Vergouwen 1991, Auharek & França 2010). SC proliferation is mainly regulated by FSH and androgens (Heckert & Griswold 2002, Sharpe et al. 2003, De Gendt et al. 2004, Johnston et al. 2004, Meachem et al. 2005, Scott et al. 2007, 2008, Auharek et al. 2011, 2012), while its maturation occurs under thyroid hormone influence, especially via p21 and p27 and via activation of the T3 receptor TRalpha1 present in these cells (Buzzard et al. 2003, Holsberger et al. 2005, Holsberger & Cooke 2005, Wagner et al. 2008, Fumel et al. 2012). When SC stops dividing and differentiate, they acquire mature characteristics, such as the development of the blood–testis barrier and fluid secretion, and become the main supporters of germ cells progression through spermatogenesis. Therefore, SC number, established at this point, is the ultimate determinant of the sperm production in adulthood (Sharpe 1994, Hess & França 2007, Griswold 2015).

The goitrogen drug 6-propyl-2-thiouracil (PTU) inhibits thyroid hormone synthesis as well as the conversion of thyroxine (T4) to tri-iodothyronine (T3), and is used experimentally to induce hypothyroidism in several mammalian species (Cooke 1991, 1992, 1993, Joyce et al. 1993, Hess et al. 1993, Cooke et al. 1994, França et al. 1995, Kirby et al. 1996, Jansen et al. 2007, Auharek & França 2010). In laboratory rodents, lower concentrations of thyroid hormones extends the SC proliferative period, delaying their transition from the mitotic/proliferative to the mature/differentiated phase. This leads to an expansion in the adult SC population and, consequently, an increase in sperm production and testis weight (Van Haaster et al. 1992, Joyce et al. 1993, França et al. 1995, Cooke 1996, Auharek & França 2010, Kobayashi et al. 2014).

In mammalian tissues, nitric oxide (NO) plays a crucial role in several physiological and pathological conditions, and is produced by nitric oxide synthase (NOS) via catalysis of l-arginine (Moncada et al. 1991, Thippeswamy et al. 2006). Different isoforms of NOS (neuronal-nNOS; endothelial-eNOS; inducible-iNOS) are expressed in the testis, and they all seem to be important for the normal function of this organ. Specifically, the iNOS isoform is constitutively expressed in Sertoli, Leydig (LC) and germ cells, including spermatozoa (O’Bryan et al. 2000, Kon et al. 2002, Lue et al. 2003, Lee & Cheng 2004, Kolasa et al. 2009). The NO has been proposed as a negative modulator of testosterone production in LC (Adams et al. 1994, O’Bryan 2000, Drewett et al. 2002, Weissman et al. 2005, Lal & Dubey 2013). In this context, we have previously demonstrated that iNOS-deficient male mice have increased anogenital distance during postnatal life and in adulthood, concomitantly with a significantly increased number and proliferation indexes of SC and LC, suggesting that higher androgen levels, especially during foetal life, could be the main factor involved in these changes. In this regard, due to higher SC proliferation and much less germ cell apoptosis, testis weight and daily sperm production (DSP) are significantly increased in iNOS−/− mice (Lue et al. 2003, Auharek et al. 2011, 2012).

Therefore, this study evaluated the effects of transient hypothyroidism induced by PTU treatment during early postnatal life in iNOS−/− mice. Specifically, we sought to determine if there is an additive effect of both conditions on SC proliferation, and the correlation of this with spermatogenesis and consequently sperm production in adulthood.

Materials and methods

Animals and treatment

The wild type (WT) (C57BCL/6) and iNOS-deficient mice (iNOS−/−; B6.129P2-Nos2tm1Lau/J, stock number 002609, purchased from the Jackson Laboratory) were housed in a standard animal facility under controlled temperature (22°C) and photoperiod (12 h light, 12 h darkness) with access to water and rodent food ad libitum.All procedures and protocols followed approved guidelines for the ethical treatment of animals according to the Ethics Committee in Animal Experimentation from the Federal University of Minas Gerais (CETEA/UFMG – Protocol #48/2010).

Hypothyroidism was induced by adding PTU (6-propyl-2-thiouracil; Sigma-Aldrich) to the mother’s drinking water, beginning the morning after parturition, in dosages of 0.006, 0.012 and 0.036% (w/v; Cooke et al. 1993, Joyce et al. 1993). To enhance palatability, a commercial sweetener (Zero-Cal) was added to the PTU-containing water. The pups received PTU through the mother’s milk from birth to 20 days of age, while control mothers/pups received tap water. After weaning, all groups received tap water ad libitum until 70 days of age, when they were sampled. Four groups were established: (I) euthyroid WT (WT, n = 9); (II) hypothyroid WT (WT + PTU, n = 9); (III) euthyroid iNOS−/− (iNOS−/− , n = 9); and (IV) hypothyroid iNOS−/− (iNOS−/− + PTU, n = 9). It should be stressed that, after several attempts, no iNOS−/− pup survived when the PTU dosage was above 0.036% (w/v). Therefore, we tested three different dosages (0.006, 0.012 and 0.036%; w/v), based on the literature (Cooke et al. 1993), and all of them were very effective in inducing hypothyroidism. In this regard, we randomly selected 3 male mice from each of the three dosages tested, obtaining 9 individuals in each treated group, which we used for the histomorphometric analysis. At sacrifice, all animals received an intraperitoneal injection of heparin (Liquemine, Roche; 125 IU/kg) and pento-barbital (Thiopentax; 150 mg/kg), and testes were perfusion fixed with 4% buffered glutaraldehyde for 20–25 min. After fixation, testes were trimmed, weighed, embebbed in plastic (glycol methacrylate) and prepared for histological and stereological evaluation.

Testis stereology

Volume densities of testicular tissue components were determined on images captured by light microscopy, using a 540-intersection grid from ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). Fifteen randomly chosen fields/images (8100 points in total) were scored for each animal at 400× magnification. The testicular components were classified as one of the following: seminiferous epithelium, tunica propria, lumen, LC, connective tissue, blood and lymphatic vessels. The volume of each testis component was determined as the product of its volume density and testis volume. For subsequent morphometric calculations, the specific gravity of testis tissue was considered to be 1.0 (França & Godinho 2003, Leal & França 2006). To obtain a more precise measure of testis parenchymal volume, the testis capsule was excluded from the testis weight.

Seminiferous tubules diameter and seminiferous epithelium height were measured at 400× magnification using an ocular micrometer calibrated with a stage micrometer. Thirty round or nearly round tubular profiles were chosen randomly and measured for each animal. The total length of the seminiferous tubule (in metres) was obtained by dividing the seminiferous tubule volume by the square radius of the tubule multiplied by π (Johnson & Neaves 1981, França & Godinho 2003, Auharek et al. 2011).

Frequencies of the stages of the seminiferous epithelium cycle

In order to evaluate spermatogenesis, stages of the cycle in mice were characterized based on the acrosomic system method, according to Russell and coworkers (Russell et al. 1990). The relative stage frequencies were determined from the analysis of approximately 200 seminiferous tubule cross-sections per animal, at 1000× magnification, as described by Leal and França (2006). Both testes were analysed for each animal. The histological sections used were those that presented better quality and more tubular cross-sections.

Cell counts and cell numbers

Pachytene primary spermatocytes, round spermatids and SC nucleoli present at the stage VII of the cycle were counted in ten randomly chosen round seminiferous tubule cross-sections for each animal. These counts were corrected for section thickness (4 µm) and nuclear or nucleolar diameter according to Abercrombie (1946), as modified by Amann and Almquist (1962). For this purpose, the diameters of ten nuclei or nucleoli were measured per animal for each cell type analysed. Cell ratios were obtained from the corrected counts. The total number of SC was determined from the corrected counts of SC nucleoli per seminiferous tubule cross-sections and the total length of seminiferous tubules according to Hochereau-de Reviers and Lincoln (1978). Daily sperm production per testis and per gram of testis was determined according to the formula developed by França (1992), as follows: DSP = total number of SC per testis x ratio of round spermatids to SC at stage VII × stage VII relative frequency (%)/stage VII duration (days).

The volume of individual LC was obtained from the nuclear and cytoplasmic volume per testis and from the number of LC. As the LC nucleus in mice is spherical, its volume was determined from its mean nuclear diameter. For this purpose, 30 nuclei showing a prominent nucleolus were measured for each animal. LC nuclear volume was expressed in µm3 and obtained by the formula 4/3πr3, where r = nuclear diameter/2. To calculate relative nuclear and cytoplasmic volumes, a 441-point square lattice was placed over the sectioned material at 1000× magnification, and 1000 points over LC were counted for each animal. The number of LC per testis was estimated from the LC individual volume and the volume density of LC in the testis parenchyma.

Statistical analysis

Values are expressed as mean ± s.e.m. and data were analysed using one-way ANOVA followed by the Bonferroni post-test, using GraphPad Prism 6 (GraphPad Software).

Results

Biometric data and testis histology

Body and testicular weights and gonadosomatic index for all four investigated groups are shown in Fig. 1 and Table 1. In comparison to all other groups, body weights of PTU-treated WT mice were significantly reduced (Table 1). Regarding the testis weight, all experimental groups showed an increase (P < 0.05) when compared to the control group. However, the iNOS−/− and iNOS−/− + PTU testis weight were similar, being significantly higher than the WT + PTU group (Fig. 1A). The same pattern was observed for the gonadosomatic index (testis mass/body weight) (Fig. 1B). Figure 2 shows that the testis parenchyma appeared normal, and with no histological alterations in all evaluated groups.

Figure 1
Figure 1

Testis weight (A) and gonadosomatic index (B) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Note that both parameters were significantly increased in the iNOS−/− groups, but no difference was found after PTU treatment. Additionally, for both parameters evaluated the WT group treated with PTU situated in an intermediate level between WT and iNOS−/− mice. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Figure 2
Figure 2

Seminiferous tubule cross-sections in wild-type (WT) and iNOS-deficient mice (−/−), treated (B and D) or not (A and C) with PTU. Observe that in all groups investigated the testis structure evaluated histologically looked normal in both compartments (tubular, ST; and intertubular, IT) of the testis parenchyma. Bar = 100 µm.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Table 1

Biometric and morphometric data in wild-type (WT) and iNOS-deficient mice (iNOS−/−), treated or not with PTU (mean ± s.e.m.).

WTWT + PTUiNOS−/−iNOS−/− + PTU
Body weight (g)26 ± 0.5a23 ± 0.8b26 ± 0.4a26 ± 0.8ab
Testis weight (mg)93 ± 2a109 ± 3b159 ± 3c152 ± 6c
Gonadosomatic index (%)0.72 ± 0.01a0.95 ± 0.03b1.22 ± 0.02c1.19 ± 0.03c
Volumetric density (%)
 Seminiferous tubules94.5 ± 0.494.7 ± 0.395.7 ± 0.395.1 ± 0.4
  Tunica propria3.1 ± 0.1a4.3 ± 0.2b3.0 ± 0.1a3.4 ± 0.2a
  Seminiferous epithelium84.5 ± 0.684.5 ± 0.581.5 ± 1.282.7 ± 1.1
  Lumen6.8 ± 0.6a5.8 ± 0.6a11.2 ± 1.5b9.0 ± 1.5ab
 Intertubular compartment5.5 ± 0.35.3 ±0.34.3 ± 0.44.9 ± 0.4
  Leydig cell3.2 ± 0.2a3.1 ± 0.3a2.0 ± 0.2b2.2 ± 0.2b
  Blood vessels1.6 ± 0.11.3 ± 0.11.9 ± 0.21.9 ± 0.2
  Lymphatic space0.25 ± 0.050.28 ± 0.070.28 ± 0.060.36 ± 0.06
  Connective tissue0.26 ± 0.06a0.59 ± 0.06b0.17 ±0.06a0.4 ± 0.10ab
Tubular diameter (µm)223 ± 3a202 ± 4b236 ± 2c223 ± 3a
Seminiferous epithelium height (µm)82 ± 275 ± 278 ± 275 ± 3
Total length of seminiferous tubule per testis (m)2.2 ± 0.1a3.1 ± 0.1b3.5 ± 0.1c3.7 ± 0.1c
Length of seminiferous tubule per testis gram (m)24.2 ± 0.5a29.8 ± 1.0b21.9 ± 0.4a24.4 ± 0.7a

Volume densities of testicular components

The majority of the testicular components evaluated showed no significant difference in volume density (Table 1). The tunica propria occupied a greater volume of the testis in the WT + PTU group (4.3%) when compared to the other three investigated groups (3.0–3.4%), while the lumen volume density was significantly higher in the iNOS−/−, when compared to the WT and WT + PTU groups. In the intertubular compartment, the volume density occupied by the LCs were significantly reduced in the iNOS−/− and iNOS−/− + PTU groups (2.0–2.2%), when compared to the WT and WT + PTU groups (3.1–3.2%). Also, in comparison to WT and iNOS−/− groups, a significant increase was observed for the connective tissue volume density in the WT + PTU mice.

Seminiferous tubules morphometry and stages frequencies

The diameter of the seminiferous tubules was similar in the WT and iNOS−/− + PTU groups, but reduced by 9.4% (P < 0.05) in the WT + PTU group and significantly increased by 5.8% (P < 0.05) in the iNOS−/− group (Fig. 3A and Table 1). The seminiferous epithelium height showed no difference between the four groups analysed (Table 1). Regarding the total length of the seminiferous tubules per testis (Fig. 3B), it was significantly increased in all experimental groups when compared to the control, being similar in the iNOS−/− and iNOS + PTU−/− , following therefore the same pattern observed for the testis weight and gonadosomatic index. Regarding the length of seminiferous tubules per testis gram, a higher value was found for the WT + PTU. Figure 4 shows that no difference was observed in the frequency of the stages of the seminiferous epithelium cycle in the four groups analysed.

Figure 3
Figure 3

Tubular diameter (A) and total length of seminiferous tubules per testis (B) in wild type (WT) and iNOS deficient mice (−/−), treated or not with PTU. Note that, in comparison to the two other groups, the results found for the tubular diameter in the WT + PTU and the iNOS−/− groups were significantly reduced and increased, respectively. In contrast, the length of the seminiferous tubules followed the same pattern observed for the testis weight and gonadosomatic index, being higher than WT in all other groups, but with no significant difference between both iNOS−/− groups. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Figure 4
Figure 4

Frequency of the stages of the seminiferous epithelium cycle in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Observe that, among the four groups analysed, no significant difference was found for any of the XII stages characterized. Data were analysed using one-way ANOVA followed by the Bonferroni post-test.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Cell counts and daily sperm production

The cell counts, ratios and DSP are shown in Table 2. The number of SC nucleoli per stage VII cross-sections was not significantly different among the four groups, while the pachytene spermatocytes number was significantly reduced (P < 0.05) in the WT + PTU group in comparison to the iNOS−/−. The round spermatids number was significantly increased in the iNOS−/− group in comparison to all other groups. Using these data, the following cell ratios were calculated: the number of round spermatids per Sertoli cell (SC efficiency); and the number of round spermatids produced per primary pachytene spermatocyte (meiotic index). Both indexes were lower in the WT + PTU-treated mice, being significant (P < 0.05) only when compared to the iNOS−/− group (Fig. 5A and Table 2).

Figure 5
Figure 5

Sertoli cell efficiency (A), total number of Sertoli cells per testis (B), daily sperm production per testis (C) and daily sperm production per testis gram (spermatogenic efficiency; D) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Note that both total number of Sertoli cells per testis and daily sperm production per testis followed the pattern already observed for testis weight and gonadosomatic index. However, the Sertoli cell efficiency was significantly reduced in the WT + PTU group only when compared to the iNOS−/− group. No significant difference among groups was found for spermatogenic efficiency. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Table 2

Cell counts and ratios per seminiferous tubule cross sections at stage VII of the seminiferous epithelium cycle, in wild-type (WT) and iNOS−/− mice, treated or not with PTU (mean ± s.e.m.).

WTWT + PTUiNOS−/−iNOS−/− + PTU
Sertoli cell nucleoli7.4 ± 0.37.8 ± 0.38.1 ± 0.37.9 ± 0.4
Pachytene spermatocytes27 ± 0.8ab25 ± 0.7a29 ± 0.6b28 ± 1.2ab
Round spermatids62 ± 1.8a54 ± 1.5a70 ± 1.3b62 ± 2.7a
Sertoli cell efficiency8.4 ± 0.4ab7.0 ± 0.2a8.7 ± 0.3b8.0 ± 0.5ab
Meiotic index2.3 ± 0.03ab2.1 ± 0.1a2.4 ± 0.04b2.2 ± 0.04ab
Number of Sertoli cell per testis (×106)3.4 ± 0.1a5.7 ± 0.2b6.8 ± 0.3c7.6 ± 0.5c
Number of Sertoli cell per testis gram (×106)37.7 ± 1.7a54.8 ± 2.5b42.9 ± 2.1ac50.2 ± 3.5bc
Daily sperm production per testis (×106)3.3 ± 0.2a4.6 ± 0.15b6.6 ± 0.3c6.8 ± 0.4c
Daily sperm production per testis gram (×106)36 ± 2.244.5 ± 2.442.2 ± 2.646.2 ± 2.5

The total number of SC per testis was increased by approximately 70% in the WT + PTU, 100% in the iNOS−/− and by 125% in the iNOS−/− + PTU groups, with no significant difference been found between the last two groups (Fig. 5B). Regarding the DSP per testis, it followed a similar pattern, being increased by 40% in the WT + PTU, 100% in the iNOS−/− and by 110% in the iNOS−/− + PTU groups (Fig. 5C). The DSP per testis gram showed no statistical difference between the four groups analysed (Fig. 5D and Table 2).

The LC morphometry is shown on Fig. 6 and Table 3. No significant difference was observed for the nuclear diameter and, consequently, for nuclear volume. The cytoplasmatic volume was lower in the iNOS−/− group, but statistically significant only when compared to the WT group. The same pattern was observed for the LC volume (Fig. 6A). In contrast, when compared to the WT, the total number of LC per testis was significantly higher in the iNOS−/− group (Fig. 6B), while no significant difference was observed for the LC number per gram of testis.

Figure 6
Figure 6

Leydig cell volume (A) and total Leydig cell number per testis (B) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Significant differences were observed for both parameters only when the control group was compared to the iNOS−/− group, being similar between the treated groups and their controls. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

Citation: Reproduction 154, 1; 10.1530/REP-17-0111

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Table 3

Leydig cell morphometry in wild-type and iNOS−/− mice, treated or not with PTU (mean ± s.e.m.).

WTWT + PTUiNOS−/−iNOS−/− + PTU
Nuclear diameter (µm)7.3 ± 0.057.3 ± 0.167.1 ± 0.067.4 ± 0.22
Leydig cell volume (µm3)993 ± 53a845 ± 62ab710 ± 29b828 ± 85ab
Nuclear volume (µm3)205 ± 4209 ± 14190 ± 5218 ± 20
Cytoplasmatic volume (µm3)787 ± 52a636 ± 49ab519 ± 24b610 ± 80ab
Leydig cell number per testis (×106)2.7 ± 0.2a3.5 ± 0.3ab4.4 ± 0.5b4.0 ± 0.6ab
Leydig cell number per testis gram (×106)29 ± 2.134 ± 3.529 ± 3.227 ± 4.1

Discussion

Understanding the mechanisms involved in SC proliferation and differentiation is critical for development of strategies related to treatment of somatic cell disorders and infertility, allowing also the discovery of potential targets for male contraception and for increasing the reproductive efficiency in economically important species. These studies are also important for increasing the knowledge of testis development and the regulation of testis function. In the present study, as expected from the literature and confirming the efficiency of the PTU treatment and the effects of iNOS deficiency in mice testis, both WT + PTU and the iNOS−/− experimental groups presented a significant increase in testis weight, gonadosomatic index, SC numbers and DSP, when compared to the wild-type group (Joyce et al. 1993, Lue et al. 2003, Auharek & França 2010, Auharek et al. 2011, 2012). However, unexpectedly, no additive effects were observed for these key parameters in the iNOS-deficient mice treated with PTU. The results herein obtained for several important parameters were not enough to draw any plausible explanation for these intriguing findings. However, as discussed later in this section, some hypotheses may be suggested.

It is worth herein mentioning that our results confirm that propylthiouracil (PTU) treatment does not alter testis cytoarchitecture (Joyce et al. 1993, Cooke et al. 2005, Auharek & França 2010). In this regard, none of the experimental groups treated with this goitrogen drug presented evident histological alteration, which can be also inferred by the unchanged volumetric density of the majority of the evaluated testis components. In addition, the frequencies of the stages of the seminiferous epithelium cycle were unaltered, indicating that the progression of the cellular associations through spermatogenesis was normal.

The tubular diameter is significantly increased in the iNOS−/− mice (Lue et al. 2003, Auharek et al. 2011), and this can be related to the higher percentage occupied by the lumen in the tubular compartment in this group. Lumen formation is resultant from fluid secretion by SC under androgen influence (Sharpe et al. 1994, Welsh et al. 2009) and it is proposed that NO may be a negative modulator of testosterone production (Adams et al. 1994, O’Bryan 2000, Drewett et al. 2002, Weissman et al. 2005, Lal & Dubey 2013), suggesting that the iNOS−/− mice may have increased androgen levels, at least in foetal/perinatal life, which can be confirmed by the increase in the anogenital distance of iNOS-deficient mice during development and adulthood, an important marker of androgen exposure during foetal life (Welsh et al. 2008, Auharek et al. 2011, 2012). In comparison to their respective controls, the tubular diameter was reduced in both groups treated with PTU. This feature may be related to a possible reduction on testosterone levels caused by the PTU treatment, which would make the iNOS + PTU phenotype similar to the WT phenotype (Hardy et al. 1993, Sarkar & Singh 2016). Also, the PTU treatment may cause a delay on mice testis development, as if the tubular diameter has not reached its full development yet, even though the mice herein analysed were sexually mature (Joyce et al. 1993, Auharek & França 2010).

The total length of seminiferous tubules is an excellent indicator of the magnitude of SC proliferation during the pre-puberal period (Hess et al. 1993, Sharpe 1994, Rey 1999, França et al. 2000). Corroborating this assumption, no additive effect was also observed in the iNOS−/− mice treated with PTU. In another point that could be considered, although the PTU treatment extends the SC proliferation period in Swiss–Webster mice (Joyce et al. 1993), this may not be true for the C57BL6/J mice strain used as a background for the iNOS−/− mice (Auharek & França 2010). Another possibility is that the SCs have their proliferative capacity exhausted in the iNOS-deficient mice, so that they are not able to proliferate more than they already did in these transgenic animals. This last assumption in particular is rather difficult to be tested. Another very important aspect to be investigated is why all iNOS-deficient mice died when treated with PTU dosages higher than 0.036%. This type of finding has not been previously reported in the literature but may reflect an important interaction between thyroid hormones and nitric oxide on regulatory mechanisms, as will be discussed below.

The cell counts and ratios obtained in the present study represent an accurate way to functionally evaluate spermatogenesis and to estimate the sperm production. Also, the balance between proliferation and apoptosis plays an important role in regulating the number of germ cells in the seminiferous epithelium, especially in the spermatogonial phase, when the number of spermatogonial cells that enter the meiotic phase needs to be limited by the SC support capacity (de Rooij 1998, de Rooij & Russell 2000, Hess & França 2007). Our analyses were focused on estimating the spermatogenic and SC efficiencies, as well as the degree of apoptosis that occurs during meiosis. The number of germ cells per seminiferous tubule cross-sections at stage VII of the seminiferous epithelium cycle tended to be lower in the PTU-treated groups, in comparison to their controls. This observation may be related to the delay in the sexual development that can be caused by PTU treatment, although the mice analysed were sexually mature (Joyce et al. 1993, Auharek & França 2010). This aspect could also reflect on the SC efficiency (the number of round spermatids supported by each SC) and the meiotic index (the number of round spermatids produced per primary pachytene spermatocyte), which showed a similar pattern.

Despite its effects on SC, the PTU treatment also influences the number and function of LC. The number of LC can be increased up to 70% in PTU-treated rats, but their volume, LH receptor numbers and their steroidogenic potential are usually reduced (Hardy et al. 1993, Cooke 1996, Mendis-Handagama et al. 1998, Teerds et al. 1998, Chiao et al. 2002, Mendis-Handagama & Ariyaratne 2005, Sarkar & Singh 2016). Similarly, iNOS-deficient mice present an increase in LC proliferation and number, from the foetal period throughout adult life, while its volume tends to be reduced (Auharek et al. 2011, 2012). In the present study, no significant differences were observed in the volumetric density and nuclear volume of LC among the four groups analysed. In contrast, comparing to the control group, the cytoplasmatic and cellular volume of these cells tended to be reduced in all experimental groups, being significant only in the untreated iNOS−/− mice, and an opposite trend was observed for the LC number per testis. Although several studies are available in the literature on this matter, the effect of PTU in LC is still a controversial subject, mainly due to different treatment windows and ages analysed. Therefore, it can be inferred that, at least in the present study, the PTU treatment had no evident effect in these steroidogenic cells.

To our knowledge, there are no reports concerning the possible interaction between thyroid hormones and nitric oxide in the testis or in the reproductive system, but it is already known that these molecules are jointly involved in regulatory mechanisms in both physiological and pathological processes in different organs, such as for instance the central nervous system (Rodríguez-Arnao et al. 2003, Xu et al. 2010), heart (Fellet et al. 2004, 2006, Kisso et al. 2008, Sarati et al. 2012), liver (Fernández et al. 2005), and immune system (Barreiro Arcos et al. 2006, 2011). Both hypothyroidism and low levels of nitric oxide cause hypertension and heart failure, and are involved in vasodilation and endothelial cells secretion (Grieve et al. 1999, Taddei et al. 2003, McAllister et al. 2005a,b, Virdis et al. 2009). It was also shown in the literature that hypothyroidism causes reduction in the nitric oxide levels, and this can be compensated by sodium nitroprusside (SNP), a NO donor compound (Rodríguez-Arnao et al. 2003, Ragginer et al. 2013). Thus, it can be hypothesized that a redundant mechanism occurs in the testis when we associate hypothyroidism with iNOS deficiency, an interesting and intriguing hypothesis that still needs to be investigated.

In conclusion, the lack of additive effect found in the present study strongly suggests that the hypothyroidism and the iNOS deficiency/low levels of nitric oxide may act through similar mechanisms on SC proliferation. Therefore, besides opening a new venue for studying the regulatory mechanisms involved in testis function, our finding brings attention to the joint involvement of these important molecules in the regulation of testis development and this interaction surely deserves further investigations.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), the Foundation for Research Support of Minas Gerais (FAPEMIG) and the Coordination for the Improvement of Higher Education Personnel (CAPES).

Acknowledgements

Technical assistance from Mara Lívia dos Santos is highly appreciated.

References

 

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Figures

  • Testis weight (A) and gonadosomatic index (B) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Note that both parameters were significantly increased in the iNOS−/− groups, but no difference was found after PTU treatment. Additionally, for both parameters evaluated the WT group treated with PTU situated in an intermediate level between WT and iNOS−/− mice. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

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  • Seminiferous tubule cross-sections in wild-type (WT) and iNOS-deficient mice (−/−), treated (B and D) or not (A and C) with PTU. Observe that in all groups investigated the testis structure evaluated histologically looked normal in both compartments (tubular, ST; and intertubular, IT) of the testis parenchyma. Bar = 100 µm.

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  • Tubular diameter (A) and total length of seminiferous tubules per testis (B) in wild type (WT) and iNOS deficient mice (−/−), treated or not with PTU. Note that, in comparison to the two other groups, the results found for the tubular diameter in the WT + PTU and the iNOS−/− groups were significantly reduced and increased, respectively. In contrast, the length of the seminiferous tubules followed the same pattern observed for the testis weight and gonadosomatic index, being higher than WT in all other groups, but with no significant difference between both iNOS−/− groups. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

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  • Frequency of the stages of the seminiferous epithelium cycle in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Observe that, among the four groups analysed, no significant difference was found for any of the XII stages characterized. Data were analysed using one-way ANOVA followed by the Bonferroni post-test.

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  • Sertoli cell efficiency (A), total number of Sertoli cells per testis (B), daily sperm production per testis (C) and daily sperm production per testis gram (spermatogenic efficiency; D) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Note that both total number of Sertoli cells per testis and daily sperm production per testis followed the pattern already observed for testis weight and gonadosomatic index. However, the Sertoli cell efficiency was significantly reduced in the WT + PTU group only when compared to the iNOS−/− group. No significant difference among groups was found for spermatogenic efficiency. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

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  • Leydig cell volume (A) and total Leydig cell number per testis (B) in wild-type (WT) and iNOS-deficient mice (−/−), treated or not with PTU. Significant differences were observed for both parameters only when the control group was compared to the iNOS−/− group, being similar between the treated groups and their controls. Data were analysed using one-way ANOVA followed by the Bonferroni post-test. Different letters indicate significant differences (P < 0.05) among groups.

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