**Table1.**VariationinproteinlevelsfromMA-10Leydigcellstreatedfor1hwithvehicle(DMSO),Fsk,Fsk+AICAR.

Nuclear receptor subfamily 4 group A member 1

 ×

 ×

 ×

The level of the protein Dymeclin, a Golgi-associated protein believed to be involved in intracellular trafficking [17–19], was the most increased after 1 h of Fsk treatment (23-fold; Table 1). Similar to NR4A1, the Fsk-dependent increase of Dymeclin was prevented when AMPK was activated by AICAR (decreased by 23-fold and back to control levels). Another protein affected by both Fsk and AICAR is the Mpv17-like protein 2 (MPV17L2). MPV17L2 is required for the assembly and stability of the mitochondrial ribosome, and in its absence, protein synthesis in mitochondria is impaired, and mitochondrial DNA aggregates [20]. Contrary to NR4A1 and Dymeclin, the MPV17L2 levels were reduced by 5.35-fold in the presence of Fsk and increased by 5.23-fold by AICAR-mediated AMPK activation (Table 1).

As summarized in Table 1, in addition to Dymeclin (up by 23-fold) and NR4A1 (up by 2.4-fold), the top Fsk-induced proteins included the homologous-pairing protein 2 homolog (PSMC3IP; up by 5-fold), acyl-coenzyme A dehydrogenase family member 12 (ACAD12; up by 4-fold), and N-acetyltransferase domain containing 1 (NATD1; up by 3.6-fold). In addition to MPV17L2, the most induced proteins in the presence of AICAR (activation of AMPK) included the cyclic AMP-responsive element-binding protein 1 (CREB1; up by 18-fold), while decreased proteins, in addition to Dymeclin and NR4A1, included the Lemur serine/threonine-protein kinase 2 (LMTK2; down by 2.8-fold). These results showed that after a short 1-h treatment, the level of some proteins involved in mitochondrial function and in steroidogenesis was already altered.

#### *2.3. Identification of Proteins Differentially Phosphorylated in MA-10 Leydig Cells in Response to Fsk and AICAR*

Our quantitative analysis of the phosphoproteome of MA-10 Leydig cells revealed significant changes in the phosphorylation levels of several phosphopeptides corresponding to various proteins in response to the treatments. The treatments included DMSO (the vehicle used as the control), Fsk to increase the cAMP levels mimicking LH-stimulated steroidogenesis, and Fsk+AICAR simultaneously, where AICAR activates AMPK, a kinase previously reported to potently inhibit hormone-induced steroidogenesis [14]. The top 10 differentially phosphorylated (up and down) phosphopeptides for each pairwise comparison (Fsk vs. DMSO, Fsk+AICAR vs. Fsk, Fsk+AICAR vs. DMSO) are presented in Table 2.

*Int. J. Mol. Sci.* **2022**, *23*, 12846

**Table 2.** Top 10 phosphopeptides differentially phosphorylated in MA-10 Leydig cells treated with vehicle (DMS), Fsk, or Fsk+AICAR (pair comparisons).

*Int. J. Mol. Sci.* **2022**, *23*, 12846

#### 2.3.1. Proteins Differentially Phosphorylated in Response to Fsk

We first compared the phosphopeptides between the control and Fsk-treated MA-10 Leydig cells to identify changes in protein phosphorylation during the stimulatory phase of steroidogenesis. This led to the identification of 12,125 phosphopeptides, of which 8471 were quantified. As shown in Figure 2A, the quantified phosphopeptides were mapped on a volcano plot based on the significance and the ratio between the Fsk-treated and control (DMSO) MA-10 Leydig cells. A total of 61 phosphopeptides had significantly different phosphorylation levels between these two conditions; the phosphorylation of 35 phosphopeptides was increased, while the phosphorylation of 26 phosphopeptides was decreased following the Fsk treatment. These 61 phosphopeptides correspond to 60 unique phosphoproteins and are shown along with their relative phosphorylation levels between the two conditions on the heatmap in Figure 2B.

**Figure 2.** Analysis of differentially phosphorylated proteins in MA-10 Leydig cells in response Forskolin. LC-MS/MS was performed on enriched phosphopeptides extracted and digested from MA-10 Leydig cells treated with either DMSO (control) or Forskolin (Fsk, 10 μm) for 1 h. Phosphorylated proteins were then quantified and compared between treatments. Results are the mean of three individual experiments,

each performed in triplicate. (**A**) Volcano plot comparing the level of phosphorylated proteins detected by LC-MS/MS in control and Fsk-treated MA-10 Leydig cells. Each dot represents a distinct phosphoprotein. A Log2 fold change of +/−1.17 (corresponds to 2.25-fold) with a *p*-value of 0.05 was used and is represented by the blue dotted lines. In response to Fsk, blue dots depict a decrease in phosphorylation level (two proteins are identified in that group: MAPT and PARD3), while red dots correspond to an increase in phosphorylation level (four proteins are identified in that group: SRP14, NR4A1, RAF1, AKAP1). Grey dots correspond to proteins with no significant changes in phosphorylation levels. (**B**) Heatmap of protein phosphorylation levels for differentially phosphorylated proteins between treatment groups (control DMSO vs. Fsk). The name of the protein is indicated. The scale on the right represents expression levels (Log10). Biological Process (**C**), Pathway Analysis (**D**), Cellular Component (**E**), and Molecular Function (**F**) of the differentially phosphorylated proteins are shown.

The proteins exhibiting increased phosphorylation after the Fsk treatment included the nuclear receptor NR4A1, the signal recognition particle SRP14, the protein kinase A anchoring protein AKAP1, and the serine/threonine kinase RAF1. In comparison, the proteins showing a reduction in their phosphorylation level in response to Fsk included the microtubule-associated protein tau (MAPT) and the Par-3 family cell polarity regulator protein PARD3 (Figure 2A,B). In response to Fsk in MA-10 Leydig cells, the differentially phosphorylated proteins are mostly implicated in the metabolic processes and the regulation of translation (Figure 2C). A pathway analysis revealed that these proteins are implicated mainly in the mTOR and Hippo signaling pathways, two pathways involved in metabolism (Figure 2D). As shown in Figure 2E, the differentially phosphorylated proteins are associated with various cellular components. Finally, the 60 differentially phosphorylated proteins are associated with various molecular functions, including binding to protein phosphatase, phosphatidyl inositol phosphate, and nucleic acid (Figure 2F).

#### 2.3.2. Proteins Differentially Phosphorylated in Response to AMPK Activation

The activation of AMPK with AICAR represses LH/cAMP-induced steroidogenesis but does not significantly affect the unstimulated steroidogenic cells [14]. Therefore, to identify potential target proteins of AMPK, we compared all phosphopeptides between the Fsk- and Fsk+AICAR-treated MA-10 Leydig cells. The differences between those two conditions should reflect mainly the proteins phosphorylated by AMPK. This comparison identified 12,125 phosphopeptides, of which 8581 phosphopeptides were quantified. It also revealed that the phosphorylated amino acid in cells exposed to AICAR was, by far, a serine, as shown by the sequence motif presented in Figure 3A. Quantified phosphopeptides were then mapped on a volcano plot based on the significance and the ratio between the Fsk+AICAR- and Fsk-treated cells (Figure 3B). A total of 46 phosphopeptides had significantly different phosphorylation levels between these two conditions; in the Fsk+AICAR group, the phosphorylation of 27 phosphopeptides was increased, while the phosphorylation of 19 phosphopeptides was decreased compared to the Fsk group (Figure 3B,C). These 46 phosphopeptides correspond to 44 unique phosphoproteins, as some proteins have more than one phosphorylated residue (phosphopeptide sequence). These 44 phosphoproteins are presented along with their relative phosphorylation levels between the two conditions on the heatmap presented in Figure 3C.

**Figure 3.** Analysis of differentially phosphorylated proteins in MA-10 Leydig cells treated with Forskolin or Forskolin+AICAR. LC-MS/MS was performed on enriched phosphopeptides extracted and digested

from MA-10 Leydig cells treated with Forskolin (Fsk, 10 μm) or Fsk+AICAR (10 μM and 1 mM, respectively) for 1 h. Phosphorylated proteins were then quantified and compared between treatments. Results are the mean of three individual experiments, each performed in triplicate. (**A**) Consensus sequence motif of phosphorylated peptides. The amino acid chosen is the one with the highest probability of being phosphorylated. (**B**) Volcano plot comparing the level of phosphorylated proteins detected by LC-MS/MS in Fsk-treated vs. Fsk+AICAR-treated MA-10 Leydig cells (to observe the effects of AICAR). Each dot represents a distinct phosphoprotein. A Log2 fold change of +/−1.17 (corresponds to 2.25-fold) with a *p*-value of 0.05 was used and is represented by the blue dotted lines. In response to AICAR, blue dots depict a decrease in phosphorylation level (one protein is identified in that group: NUMA1), while red dots correspond to an increase in phosphorylation level (three proteins are identified in that group: MAPT, PARD3, NFATC3). Grey dots correspond to proteins with no significant changes in phosphorylation levels. (**C**) Heatmap of protein phosphorylation levels for differentially phosphorylated proteins between treatments (Fsk vs. Fsk+AICAR). The name of the protein in indicated. The scale on the right represents expression levels (Log10). Biological Process (**D**), Pathway Analysis (**E**), Cellular Component (**F**), and Molecular Function (**G**) of the differentially phosphorylated proteins are shown.

The proteins with increased phosphorylation in the AICAR group included NFATC3 (transcription factor), PARD3, and MAPT; an example of a protein with reduced phosphorylation in the AICAR group is the nuclear mitotic apparatus protein NUMA1 (Figure 3A,B). The proteins differentially phosphorylated between the Fsk+AICAR and Fsk groups are mostly implicated in the microtubule polymerization or its organization (Figure 3D). This is supported by the localization of these proteins, mostly with microtubules (Figure 3F), with their main molecular functions being microtubule binding, histone binding, and deubiquitinase activity (Figure 3G). The pathway analysis revealed that the 44 proteins are mainly associated with the MAPK signaling pathway and mRNA surveillance pathway (Figure 3E).

#### 2.3.3. Proteins Differentially Phosphorylated in Response to Combined Fsk+AICAR

Comparisons were made between the Fsk+AICAR and the control (DMSO) groups to identify all the changes in the protein phosphorylation triggered by the simultaneous treatment of the MA-10 Leydig cells with Fsk (stimulatory) and AICAR (inhibitory). A total of 12,125 phosphopeptides were identified, of which 8711 were quantified. As shown in Figure 4A, the quantified phosphopeptides were mapped on a volcano plot based on the significance and the ratio between the Fsk+AICAR and control (DMSO) cells. A total of 73 phosphopeptides exhibited significantly different phosphorylation levels between these two conditions; the phosphorylation of 38 phosphopeptides was increased in the MA-10 Leydig cells treated with Fsk+AICAR, while the phosphorylation of 35 phosphopeptides was decreased. These 73 phosphopeptides correspond to 68 unique phosphoproteins, as some proteins harbor more than one phosphorylation site, such as MAP1A. The 68 phosphoproteins identified, along with their relative phosphorylation levels between the two conditions, are shown on the heatmap presented in Figure 4B.

Some of the proteins with increased phosphorylation in the MA-10 Leydig cells in response to Fsk+AICAR included NR4A1, NUMA1, the protein GOLGA5 involved in maintaining the Golgi structure, and the microtubule-associated proteins MAP1A and MAP1B. MAP1A was also present amongst the proteins with reduced phosphorylation. This is explained by the fact that MAP1A contains two distinct phosphopeptides: one showed increased phosphorylation, while the other showed reduced phosphorylation in the presence of Fsk+AICAR (Figure 4A). In terms of the biological processes, differentially phosphorylated proteins were mainly associated with microtubule polymerization, depolymerization, and organization (Figure 4C). Consistent with this, these proteins are mostly localized with microtubules, and their main functions are to bind to the microtubules, actin, and tubulin (Figure 4F). The pathway analysis associated the differentially phosphorylated proteins with the axon guidance pathway (Figure 4D).

Taken together, these results provide the first description of the global changes in the phosphoproteome of MA-10 steroidogenic Leydig cells after a stimulatory treatment (Fsk) and an inhibitory treatment (activation of AMPK by AICAR).

**Figure 4.** Analysis of differentially phosphorylated proteins in MA-10 Leydig cells in response to Forskolin+AICAR. LC-MS/MS was performed on enriched phosphopeptides extracted and digested

from MA-10 Leydig cells treated with DMSO (control) or Fsk+AICAR (10 μM and 1 mM, respectively) for 1 h. Phosphorylated proteins were then quantified and compared between treatments. Results are the mean of three individual experiments, each performed in triplicate. (**A**) Volcano plot comparing the level of phosphorylated proteins detected by LC-MS/MS in control (DMSO) vs. Fsk+AICARtreated MA-10 Leydig cells. Each dot represents a distinct phosphoprotein. A Log2 fold change of +/−1.17 (corresponds to 2.25-fold) with a *p*-value of 0.05 was used and is represented by the blue dotted lines. In response to Fsk+AICAR, blue dots depict a decrease in phosphorylation level (one protein is identified in that group: MAP1A), while red dots correspond to an increase in phosphorylation level (five proteins are identified in that group: MAP1A, MAP1B, NR4A1, NUMA1, GOLGA5). Grey dots correspond to proteins with no significant changes in phosphorylation levels. (**B**) Heatmap of protein phosphorylation levels for differentially phosphorylated proteins between treatments (control DMSO vs. Fsk+AICAR). The name of the protein is indicated. The scale on the right represents expression levels (Log10). Biological Process (**C**), Pathway Analysis (**D**), Cellular Component (**E**) and Molecular Function (**F**) of the differentially phosphorylated proteins are shown.

#### *2.4. Phosphorylation Trajectory of Representative Proteins*

Several proteins exhibited an altered phosphorylation status in response to Fsk and to Fsk+AICAR in the MA-10 Leydig cells (Figures 2–4). Although the protein levels remained unchanged for the majority of proteins (Table 1), changes in the phosphorylation level in response to Fsk and to Fsk+AICAR could still be the result of a concomitant change in the protein level. Therefore, the phosphorylation status of the representative proteins was plotted and compared to their protein levels, allowing for better visualization of the phosphorylation trajectory of a given protein.

The phosphorylation level of NR4A1, an orphan nuclear receptor known to regulate the expression of several steroidogenic genes, was increased by more than 20-fold after the Fsk treatment, while the total NR4A1 protein level was only increased by 2.4-fold. This increase tended to be attenuated when cells were treated with AICAR in addition to Fsk (Figure 5A). Interestingly, the phosphorylation levels of the progesterone membrane receptor components 2 (PGRMC2), a protein belonging to the membrane-associated progesterone receptor (MAPR) protein family, was significantly increased when cells were treated with Fsk and with Fsk+AICAR (Figure 5B). No change in the total PGRMC2 protein level was noted. The NUMA1 exhibited the same phosphorylation pattern as PGRMC2 (Figure 5C), with an increase in phosphorylation of the phosphopeptide QAASSQEPSELEELR. On the other hand, a different phosphopeptide (IASSSSENNFLSGSPSSPMGDILQTPQFQMR) of the NUMA1 protein remained unchanged in response to Fsk, whereas AICAR caused a significant reduction in its phosphorylation level (Figure 5D). Finally, the phosphorylation level of two proteins, known to be downstream of AMPK, MAPT, and PARD3, was significantly reduced when the MA-10 Leydig cells were treated with Fsk. However, this decrease in phosphorylation was no longer apparent when the cells were co-treated with Fsk+AICAR, suggesting that AMPK phosphorylates these proteins (Figure 5E,F). The total protein level of PGRMC2, NUMA1, MAPT, and PARD3 was not affected by the treatments. These results validate AMPK activation in the MA-10 Leydig cells treated with AICAR.

Altogether, these results reveal that protein phosphorylation, in response to Fsk and Fsk+AICAR in MA-10 Leydig cells, is a very dynamic process and is mainly independent of the changes in protein levels.

**Figure 5.** Representative proteins that are differentially phosphorylated in MA-10 Leydig cells in response to Forskolin and to Forskolin+AICAR. Intensity plots of representative phosphoproteins (blue lines) and total proteins (red lines). Ctl, DMSO-treated cells; Fsk, Forskolin-treated cells; Fsk+AICAR, cells treated with Forskolin and AICAR. Results are shown as Fold Change compared to the control. Results are the mean of three individual experiments +/−SEM. The phosphopeptide sequence is shown beneath the graph. For a given protein, different letters indicate a statistically significant difference between treatment groups (*p* < 0.05).

#### **3. Discussion**

Several studies have reported changes in the expression of a single gene or its protein levels in response to hormone-induced steroidogenesis. However, Leydig cell steroidogenesis is a strictly regulated process that requires changes in gene expression, protein levels, and protein phosphorylation of many genes and proteins. Therefore, the main objective of this work was to elucidate the global phosphoproteomic changes that occur in MA-10 Leydig cells in response to Forskolin-induced steroidogenesis, followed by AICAR-activated AMPK, leading to repression of steroidogenesis.

#### *3.1. Global Variation in Protein Levels in MA-10 Leydig Cells 1 h after Fsk or Fsk+AICAR Treatment*

We first determined the changes in the global protein levels and found that the level of only 20 proteins was significantly modified in the Leydig cells treated for 1 h with either Forskolin alone (stimulatory) or Forskolin+AICAR (inhibitory). One of those proteins was the nuclear receptor NR4A1. Our global proteomic analysis revealed that the NR4A1 protein levels were increased by 2.4-fold after the Forskolin treatment, and this increase was blunted when cells were co-treated with AICAR. This is consistent with previous studies from our group that reported the pattern of *Nr4a1* mRNA levels [14], as well as a time course of NR4A1 protein levels [10], in response to (Bu)2-cAMP treatment of mouse MA-10 Leydig cells and primary Leydig cells from rats. Therefore, the observed changes in the NR4A1 protein levels in our current study validate our experimental approach.

Another protein affected by the treatments was Dymeclin, which was induced 23-fold in response to Fsk and then lost upon AMPK activation. Although it has never been reported in Leydig cells, Dymeclin is believed to be implicated in vesicle trafficking, as it shuttles between the cytosol and the Golgi apparatus in a highly dynamic manner [18,19]. Vesicle trafficking is an important process for steroidogenesis. For instance, the life cycle of lipid droplets, which are a source of cholesterol substrate for steroid hormones [21], involves intracellular trafficking (reviewed in [22]). The significant and rapid changes in Dymeclin protein levels in response to Fsk and Fsk+AICAR suggest that Dymeclin might play an active role in hormone-regulated steroidogenesis in Leydig cells.

The transcription factor, CREB, is known to activate steroidogenic gene expression in Leydig cells [23–28]. Despite this, the CREB protein levels remained unchanged in response to the Fsk/cAMP treatment, which is identical to what we observed in our current global proteomic analysis. Unexpectedly, the CREB protein levels were increased by 18-fold in the MA-10 Leydig cells treated with AICAR to turn on the repressive kinase, AMPK (Fsk+AICAR-treated MA-10 Leydig cells compared to the Fsk-treated cells). This would appear to suggest a repressive role for CREB in steroidogenesis. Although mainly known as an activator, CREB has also been reported to act as a repressor. For instance, CREB represses the expression of the *cFos* gene [29]. Furthermore, alternate exon usage is also known to switch CREB from an activator to a repressor [30]. It is, therefore, possible that activated AMPK influences CREB exon usage and that CREB would, therefore, repress steroidogenic gene expression in that context.

The MPV17L2 protein levels were also modulated by the treatments. MPV17L2 is an integral mitochondrial inner membrane protein [20], and as such, it is implicated in mitochondrial ribosome assembly [20] and in reactive oxygen species (ROS) production [31], two processes known to influence steroidogenesis in Leydig cells [32]. We found that the MPV17L2 protein levels increased 5-fold in the presence of AICAR, while it was reduced by 5-fold in the Fsk-treated cells. Our data are consistent with transcriptomic data from the pancreas, where the *Mpv17l2* mRNA levels are decreased by 1.6-fold in AMPK-deficient mice [33], indicating that activated AMPK increases its expression.

Overall, our data show that globally, the levels of 20 proteins, including some never before reported in Leydig cells, are rapidly and differentially modulated in response to Fsk (stimulatory) and to Fsk+AICAR (inhibitory).

#### *3.2. Comprehensive Analysis of the Phosphoproteome during Stimulation and Inhibition of Steroidogenesis in MA-10 Leydig Cells*

In Leydig cells, the response to hormone stimulation involves the activation of various signaling pathways, leading to increased activity of various kinases, including PKA, MAPK, CAMKI, and AMPK. These kinases then phosphorylate various proteins, thus ensuring a proper cellular response to the stimulus. Although some phosphorylated proteins have been identified, the majority of studies have focused on a single protein at the time. One study reported the changes in the phosphoproteome of Leydig cells in which the cAMP levels were maintained artificially high by inhibiting all phosphodiesterase activity [34]. This study identified alterations in several phosphosites, some of which were also detected in our current study. However, constitutively high cAMP levels might trigger non-physiological responses. In our present work, we used Fsk, an agonist of adenylate cyclase that leads to a typical increase in cAMP levels, triggering a stimulatory response, and AICAR, an agonist of AMPK, leading to an inhibitory response. In both cases, the levels of cAMP, the main second messenger in Leydig cells, followed a normal increase–decrease rate typical of a more classic response [14]. Using this treatment scheme and a quantitative phosphoproteomics approach, we now report the global changes in the phosphoproteome that occur in Leydig cells in response to both a stimulatory and an inhibitory signal. We found that the phosphorylation level of a large number of proteins was modified in response to either Fsk (stimulatory response) or Fsk+AICAR (inhibitory response).

The proteins displaying increased phosphorylation levels in the MA-10 Leydig cells treated with Fsk include AKAP1 (also known as AKAP121), RAF1, NR4A1, and SRP14. The phosphorylation of both AKAP1 and RAF1 was previously reported as being altered in the MA-10 Leydig cells depleted of PDE4 and PDE8 activity [34]. PDE activity is required for normal Leydig cell function. Indeed, PDEs regulate steroidogenesis by their ability to degrade cAMP into AMP, thus inactivating several cAMP-dependent pathways and kinases, such as PKA, while simultaneously activating the repressive kinase AMPK [12]. AKAP1 is a member of the AKAP family of scaffold proteins. AKAP1 is known to interact with PKA and therefore regulates its intracellular localization, especially at the outer mitochondrial membrane (OMM) (reviewed in [35]), where PKA can phosphorylate its target proteins [36]. A key target of PKA at the OMM for the stimulation of steroidogenesis is the STAR protein [36,37]. PKA-dependent phosphorylation of STAR increases its cholesterol shuttling activity, leading to enhanced steroidogenesis [36,37]. AKAP1 is, therefore, considered an important mitochondrial signaling hub, and the mitochondrion is an essential organelle for steroidogenesis in Leydig cells. To date, AKAP1 is not known to be phosphorylated. Our current work demonstrates that AKAP1 is phosphorylated in response to Fsk. The physiological implication of AKAP1 phosphorylation remains to be established, although a previous study suggested that phosphorylation of AKAP1 can modulate the AKAP1-PKA association [38].

Another protein exhibiting increased phosphorylation in response to Fsk is SRP14. SRP14 is involved in protein translation and, more specifically, in the elongation arrest and efficient translocation of proteins into the endoplasmic reticulum [39]. This is consistent with the requirement of a de novo protein synthesis in hormone-induced steroidogenic cells [10]. Interestingly, the level of SRP14 protein in glioblastoma cells is increased by CYP17A1 [40], an enzyme implicated in steroidogenesis. As we observed in the Fsktreated MA-10 Leydig cells, SRP14 was also found to be phosphorylated during a large screen of phosphorylated proteins in Ras-transformed cells [41]. The significance of SRP14 phosphorylation, however, remains unknown.

The proteins displaying reduced phosphorylation levels after Fsk treatment include MAPT and PARD3. Phosphorylation of both proteins was restored when AMPK was activated (Fsk+AICAR-treated cells). MAPT, a protein involved in microtubule assembly and stabilization, is a known target of AMPK [42]. Furthermore, phosphorylation of MAPT is reduced in the MA-10 Leydig cells deficient in PDE4 and PDE8 activity to maintain high cAMP levels [34]. These data are consistent with our present data showing a reduction in MAPT phosphorylation in the context of high cAMP levels (Fsk-treated cells) and increased phosphorylation in AMPK-activated cells. Phosphorylation of MAPT is known to influence protein stability and degradation (reviewed in [43]).

The protein PARD3 is part of the PAR3/PAR6/aPKC complex implicated in tightjunction assembly [44]. Activation of AMPK facilitates tight-junction assembly [45]. Whether activated AMPK interacts with and directly phosphorylates the PAR3/PAR6/aPKC complex remains uncertain [45]. However, our current data now indicate that PARD3 is phosphorylated upon AMPK activation. Furthermore, PARD3 is known to be phosphorylated by the PAR1 kinase, leading to PARD3 translocation from the tight junction to the cytosol [46]. Once in the cytosol, PARD3 promotes the interaction between PP1A phosphatase and LATS1, a mediator of the Hippo signaling pathway [46]. This results in the dephosphorylation and inactivation of LATS1 and, consequently, dephosphorylation, activation, and nuclear translocation of the TAZ transcriptional co-activator [46]. Once in the nucleus, TAZ represses Leydig cell steroidogenesis by suppressing the expression of several steroidogenic genes via an interaction with the nuclear receptor NR4A1, preventing it from binding to DNA and activating its target genes [47]. It is tempting to speculate that the phosphorylation of PARD3, which we observed in the AMPK-activated Leydig cells, might lead to the same events, therefore contributing to a repression of steroidogenesis.

The transcription factors belonging to the nuclear factor of activated T cells (NFAT) family are well-known transcriptional activators (reviewed in [48,49]). In our phosphoproteomic screen, the phosphorylation of the transcription factor NFATC3 remained unchanged in the presence of Fsk alone. However, it was significantly increased in the MA-10 Leydig cells treated with Fsk+AICAR, indicating that NFATC3 becomes phosphorylated upon activation of AMPK. Interestingly, phosphorylated NFAT transcription factors are known to be sequestered in the cytoplasm since phosphorylation masks the nuclear localization sequence [50,51]. The phosphorylation of NFATC3 that occurs upon activation of AMPK in MA-10 Leydig cells would be consistent with the exclusion of NFATC3 from the nucleus and a reduction in its ability to activate gene expression, therefore contributing to reduced steroidogenesis.

Another group of differentially phosphorylated proteins includes those where the phosphorylation level was significantly different only in the Fsk+AICAR-treated MA-10 Leydig cells vs. control cells (vehicle-treated). This included the NUMA1 protein, which was differentially phosphorylated on two different peptides; one exhibited increased phosphorylation, while the phosphorylation of another residue of the protein was significantly decreased. NUMA1 is a component of the nuclear matrix (reviewed in [52]) and is implicated in ciliogenesis and autophagy, as is AMPK [53]. NUMA1 was indeed proposed as a potential target of AMPK, although this remains to be validated [54]. Our current results support direct phosphorylation of NUMA1 by AMPK.

The PGRMC2 protein also exhibited increased phosphorylation in the MA-10 Leydig cells treated with Fsk+AICAR vs. controls. PGRMC2 is a membrane-associated progesterone receptor (MAPR) present in Leydig cells and is believed to play a role in the progesterone auto/paracrine action on Leydig cells [55,56] in addition to potentially regulating the activity of some cytochrome P450 enzymes [57]. We showed that PGRMC2 is present in MA-10 Leydig cells and that it is phosphorylated after AMPK activation.

The MA-10 Leydig cell line is the gold standard for studying Leydig cells, although they are not identical to the cultured primary Leydig cells. The MA-10 Leydig cell line was originally established from a Leydig cell tumor in a mouse (M5480P) [58] and has since been meticulously characterized. Treatment of MA-10 cells with the luteinizing hormone, Forskolin, or cAMP analogs, increases steroid hormone production in the same way as in normal Leydig cells ([58] and reviewed in [59]). The rate-limiting step in hormone-induced steroidogenesis is the transport of cholesterol into the mitochondria, where steroidogenesis is initiated (reviewed in [60]); this is also true for MA-10 Leydig cells [10,15,61]. MA-10 cells mainly produce progesterone because of a mutation in the *Cyp17a1* coding sequence [58]. MA-10 Leydig cells are, therefore, suitable for the study of hormone-induced steroidogenesis and, more

specifically, the early steps of steroidogenesis [62]. Since our current work focuses on the modulation of protein phosphorylation that occurs early following hormone treatment, the MA-10 Leydig cell line is a suitable and convenient model to use.

In conclusion, using a quantitative approach, our present study determined the global phosphoproteomic profile of MA-10 Leydig cells in response to Fsk (stimulation of steroidogenesis) and AICAR-mediated AMPK activation (repression of steroidogenesis). Our results indicate that steroidogenesis is a very dynamic process that involves differential protein phosphorylation by various kinases, altogether contributing to the fine-tuned regulation that is needed to achieve the appropriate production of steroid hormones.

#### **4. Materials and Methods**

#### *4.1. Cell Culture*

The MA-10 cell line was obtained from ATCC (Cat# CRL-3050, RRID:CVCL\_D789. ATCC, Manassas, VA, USA). The MA-10 cells were grown in a DMEM/F12 medium supplemented with 2.438 g/L sodium bicarbonate, 3.57 g/L HEPES, and 15% horse serum on gelatin-coated plates. Penicillin and streptomycin sulphate were added to the cell culture media to a final concentration of 50 mg/L, and the cells were kept at 37 ◦C, 5% CO2, in a humidified incubator. The MA-10 Leydig cell line was validated by morphology and by quantifying the progesterone output, as previously described.

#### *4.2. Chemicals*

The AMPK agonist, AICAR, was obtained from Tocris Bioscience (Minneapolis, MN, USA). Forskolin (Fsk) was purchased from Sigma-Aldrich, Canada (Oakville, ON, Canada).

#### *4.3. RNA Isolation, Reverse Transcription, and Quantitative PCR*

The MA-10 Leydig cells were cultured in the presence of either DMSO (vehicle), Fsk (10 μM), or Fsk+AICAR (10 μM + 1 mM) for 60 min. Isolation of RNA, cDNA synthesis, and reverse transcription-quantitative PCR (RT-qPCR) were performed as previously described [63,64]. Briefly, the total RNA from the MA-10 Leydig cells grown and treated, as described above, was isolated using TRIZOL (Life Technologies, Burlington, ON, Canada) and reverse-transcribed using the iScript Advanced cDNA Synthesis Kit (Bio-Rad Laboratories, ON, Canada). A quantitative real-time PCR was performed using a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, ON, Canada) along with the SsoAdvanced Universal SYBR Green Supermix kit (Bio-Rad Laboratories, ON, Canada) according to the manufacturer's protocols. Relative expression was normalized to the expression of *Rpl19*, which was used as an internal control. The mouse *Star* primers were: forward 5- -GTT CCT CGC TAC GTT CAA GC-3 and reverse 5- -GAA ACA CCT TGC CCA CAT CT-3- . The mouse *Rpl19* primers were: forward 5- -CTG AAG GTC AAA GGG AAT GTG-3 and reverse 5- -GGA CAG AGT CTT GAT GAT CTC-3- . The melting temperature of both primer couples was 62.6 ◦C.

#### *4.4. Sample Preparation for LC-MS/MS*

The MA-10 Leydig cells (5.8 million) were plated in 15-cm plates and then treated at 80% confluence with either the vehicle (DMSO), Fsk (10 μM) or Fsk+AICAR (10 μM + 1 mM) for 60 min at 37 ◦C and 5% CO2. This experiment was repeated three times in triplicate.

Protein extraction, digestion, phosphopeptide enrichment, and mass spectrometry analyses were performed by the proteomics platform of the CHU de Quebec Research Centre (Quebec City, QC, Canada). Cell pellets were resuspended in an extraction buffer (50 mM ammonium bicarbonate, 0.5% sodium deoxycholate (SDC), 50 mM DTT, protease inhibitor cocktail, phosphatase inhibitor PhosSTOP, and 1 μM pepstatin) and vortexed for 1 min. They were then sonicated with a microprobe (Sonic Dismembrator 550, Fisher Scientific) 20 times for 1 s (on/off). The extract was centrifuged at 16,000× *g* for 15 min, the supernatant was collected, and then acetone-precipitated. The pellet was resuspended

in ammonium bicarbonate 50 mM/SDC 1% and treated with bioruptor 15 times for 30 s (on/off). The protein concentration was determined by performing a Bradford assay.

Protein aliquots of 10 μg for the global protein analysis or 2 mg for the phosphopeptide analysis for each condition (vehicle, Fsk, Fsk+AICAR) were used. The proteins were first heated at 95 ◦C for 5 min and reduced with 0.2 mM DTT at 37 ◦C 30 min. They were then alkylated with 0.8 mM IAA (iodoacetamide) for 30 min at 37 ◦C in the dark. This was followed by digestion with trypsin (1:50) at 37 ◦C overnight. Digestion was stopped with the addition of formic acid; the sodium deoxycholate was removed after centrifugation. The peptides were desalted using a stage-tip column (for the global protein analysis) or HLB column (for the phosphopeptide analysis).

Phosphopeptide enrichment was performed using the High Select Phosphopeptide Enrichment Kits & Reagents kit (Fisher Scientific Canada, Catalog number A32993) according to the manufacturer's protocol. Two (2) mg of dry protein from each condition (vehicle, Fsk, Fsk+AICAR) were resuspended in 150 μL of binding/equilibration buffer (pH < 3). The TiO2 spin-tip columns were washed and then equilibrated before adding 150 μL of the resuspended samples. The TiO2 spin-tip columns loaded with the samples were centrifuged twice at 1000× *g* for 5 min to bind the phosphopeptides. The TiO2 spin-tip columns were then washed twice with the binding/equilibration buffer, once with the wash buffer and then with HPLC water. The phosphopeptides were finally eluted with 50 μL of phosphopeptide elution buffer twice.

#### *4.5. Quantitative Sample Analysis by LC-MS/MS*

Aliquots of 1 μg (for global protein analysis) and 1 mg of enriched phosphopeptide were analyzed by nanoLC-MS/MS using a Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific, Ontario, Canada), connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Ontario, Canada). Peptides were trapped at 20 μL/min in a loading solvent (2% acetonitrile, 0.05% TFA) on a 5 mm × 300 μm C18 Pepmap cartridge pre-column (Thermo Fisher Scientific, Ontario, Canada) for 5 min. The pre-column was then switched online with a Pepmap Acclaim column (Thermo Fisher Scientific, Ontario, Canada) 50 cm × 75 μm internal diameter separation column. The peptides were eluted with a linear gradient of 5–40% solvent B (A: 0.1% formic acid, B: 80% acetonitrile, 0.1% formic acid) over 270 min for a total of a 300-min run at 300 nL/min for the global protein analysis. For the phosphopeptide analysis, elution with the solvent gradient was completed in 90 min for a total run of 120 min. Mass spectra were acquired using a data-dependent acquisition mode using Thermo Xcalibur software, version 4.1.50. Full-scan mass spectra (350 to 1800 m/z) were acquired in the orbitrap using an AGC target of 4e5, a maximum injection time of 50 ms, and a resolution of 120,000. An internal calibration using lock mass on the m/z 445.12003 siloxane ion was used. Each MS scan was followed by the acquisition of fragmentation involving MS/MS spectra of the most intense ions for a total cycle time of 3 s (top speed mode). The selected ions were isolated using the quadrupole analyzer in a window of 1.6 m/z and fragmented by higher energy collision-induced dissociation (HCD) with 35% of collision energy. The resulting fragments were detected by the linear ion trap at a rapid scan rate with an AGC target of 1 × <sup>10</sup><sup>4</sup> and a maximum injection time of 50 ms. The dynamic exclusion of previously fragmented peptides was set for a period of 30 s and a tolerance of 10 ppm.

#### *4.6. Quantitative Data Analyses*

Spectra were searched against the Uniprot Reference mus musculus (61,295 entries) using the Andromeda module of the MaxQuant software, v. <IP_ADDRESS>. The trypsin/P enzyme parameter was selected with two possible missed cleavages. The carbamidomethylation of cysteines was set as a fixed modification, while methionine oxidation and phosphorylation (Ser, Thr, Tyr) were set as variable modifications. The mass search tolerances were 5 ppm and 0.5 Da for MS and MS/MS, respectively. For the protein validation, a maximum false discovery rate of 1% at the peptide and protein levels was used based on a target/decoy

search. MaxQuant was also used for label-free quantifications. The 'match between runs' option was used with a 20 min value as the alignment time window and 0.7 min as the match time window. For the global protein analysis, the protein group files were used, while for the phosphopeptide analysis, the modificationSpecifPeptides file was used. RStudio 1.2.5019 was used for data processing. A normalization step was performed using the median of the median intensities of each condition. The missing peptide intensity values were replaced by a noise value corresponding to the 1% percentile of the normalized value for each condition. A peptide was considered quantifiable only if at least three intensity values in one of the two conditions were present and with a minimum of two peptides (for the global protein analysis).

#### *4.7. Statistical Analysis*

For the RT-qPCR, statistical analyses were carried out using a nonparametric one-way ANOVA on ranks via the Kruskal–Wallis test, followed by a post-hoc Mann–Whitney U test. A *p*-value of < 0.05 was considered significant. For the LC-MS/MS analysis, a *p*-value limma test, a limma *q*-value (Benjamin–Hochberg correction), and a z-score were calculated. Phosphopeptides or proteins were considered variants if the *q*-value was <0.05 and the z-score was +/− 1.96. All statistical analyses were performed using the OriginPro Version 2021 software (www.originlab.com, accessed on 10 August 2022) (OriginLab Corporation, Northampton, MA, USA).

**Author Contributions:** Z.B.D. performed all the experiments. Z.B.D. and J.J.T. analyzed and interpreted the data. J.J.T. conceived the study and coordinated and supervised the project. Z.B.D. drafted the manuscript with the assistance of J.J.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant from the Canadian Institutes of Health Research (CIHR) (funding reference number PJT-148738) to JJT.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All mass spectrometry data (raw files and MaxQuant search result files) are publicly available on the ProteomeXchange repository (www.proteomexchange.org) with the identifier PXD037514.

**Acknowledgments:** We would like to thank Sylvie Bourassa (proteomics platform of the CHU de Quebec Research Centre, Quebec City) for her assistance with the quantitative protein and phosphoprotein mass spectrometry approach and Lisa Lahens for her assistance with the OriginPro software.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*2.3. CRU/CRUNCEP*

We used 0.5 × 0.5 degree monthly temperature and precipitation from Climate Research Unit (CRU-TS3.23) and solar radiation data from CRU National Centers for Environmental Prediction version 4 (CRUNCEP-V4, a blend of CRU data and NCEP- National Center for Atmospheric Research (NCAR) reanalysis data) [25]. CRU data are interpolated gridded datasets from monthly observations. To be consistent with historical CMIP5 runs, we used CRU/CRUNCEP data from overlapping the period with Global Inventory Modeling and Mapping Studies (GIMMS)-LAI data during 1982–2005.


**1. Introduction**

[28] Claes, M., Lacourse, E., Bouchard, C., & Perucchini, P., Parental practices in late ado‐ lescence, a comparison of three countries: Canada, France, and Italy. Journal of Ado‐

[29] Kaufmann, D., Gesten, E., Santa Lucia, R.C., Salcedo, O., Rendina-Gobioff, G., & Gadd, R. The relationship between parenting style and children's adjustment. Jour‐

[30] Chao, R.K. Beyond parental control and authoritarian parenting style: Understand‐ ing Chinese parenting through the cultural notion of training. Child Development,

[31] Park, H.S., & Bauer, S., Parenting practices, ethnicity, socioeconomic status and Aca‐ demic achievement in adolescents. School Psychology International, 2002; 23 (4),

[32] Diaz, M.V., The relations among parenting style, parent-adolescent relationship, fam‐ ily stress, cultural context and depressive symptomatology among adolescent fe‐

lescence, 2003; 26, 387-399.

96 Parenting in South American and African Contexts

1994; 65, 1111-1119.

386-396.

nal of Child and Family Studies, 2000; 9, 231-245.

males. Doctoral dissertation, Georgia state University 2005.

Parenthood is known for its importance in the development of children in different parts of the world. Parental relationships consist of routine actions of care that are necessary for the survival and well-being of the young, including beliefs, values, and expectations in a given culture [1- 6].

Through the journey of daily care, parents and caretakers gradually prepare children with education that is considered important to be inserted into various social contexts, in addition to taking care of the physical and emotional needs of children [4]. Therefore, parenthood involves social practices performed by those who take care of or are responsible for the education of children, serving as examples of values and beliefs. Culture and history also participate in this process [5,6].

What is considered adequate, as well as practices that promote what is considered appropriate in the development of children vary from culture to culture, even within the same country over time history [4]. According to the authors [6] this understanding reflects the contextualist paradigm that challenges the neo-positivist view of the data collected in a given society may be relevant in another, as well as data collected in a particular historical moment are necessarily significant in a period later [6]. In this sense, it is necessary to examine parenting in many social groups.

According to reference [7], the differences observed in beliefs and care practices in different cultures can be described by cultural models that express different methods of parenthood. Parents with high levels of education who live in a city have socialization techniques that are different from those of families with low levels of education who live in the country [7,6].

© 2013 da Silva Costa et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In this perspective, the author [3] that made extensive transcultural research, tell us about the differences between the parental practices in terms of cultural prototypes, as being those which can valorize the autonomy within the caring practices, such as situation of the American families, and others that reforce the obedience and the sense of belonging to the group, such as the case of the rural communities. The same way, significant differences are found between the practices of the Minority World (typically represented by the white middle class from North America and Western Europe) and Majority (countries with low human development index) [8]. Such differences take in consideration aspects of the point of view economic and cultural and are evidenced from points such as: the attachment mother-baby, the shared responsibility, the probability of mortality, the size of the family, the roles played by the members of the family and the surrounding community [8, 3].

Noteworthy is the fact that studies that compared educational practices between the Minori‐ tary World and poor rural communities in the Majoritary World led to a consensus that parental practices in those countries are a shortcoming [8,3,4].Therefore, parental practices in countries of the Minoritary World are considered more adequate, and such a trend is empha‐ sized by the use of measurement scales and tools for psychological investigation that advocate universal practices [5].

An important topic to evidence is the fact that the studies interested in comparing the educa‐ tional practices observed between the Minority World and the impoverished rural commun‐ ities of the Majority World have conducted in a mistaken way to the consensus that the parental practices in those countries are presented as deficient [8, 3, 4]. In this sense, the parental practices present in the countries of the Minority World are taken as more adequate which trend is to reinforce by the use of measure scales and psychological investigation instruments that profess them as universal practices [5].

**Figure 1.** The Amazonian context: the florest and housing typically riverine Amazon (collection of Laboratory Ecology

Parenting in Amazonian Riverside Context http://dx.doi.org/10.5772/57056 99

The Amazon river region consists of a forest rich in biodiversity, possessing 20% of the freshwater on the planet. Additionally, it represents 50% of the Brazilian territory and has approximately 11 million people, in which 62.4% live in the cities and 37.6% live in the country [14].The riverine area still has energetic resources and wealth and is rich with lush flora and fauna, but it is still considered an economically poor region compared with its natural richness

The riverine communities arranged in the Amazon forest are considered traditional, with occupations associated with waterways, cultures with riverine roots, and small-scale agricul‐ ture [16]. Although sharing a way of life and traditional cultures, significant differences are found among these communities in terms of access to public policies and economic develop‐ ment. Thus, riverine communities that live in geographic isolation have more financial stringency and depend more on forest resources and less on commercial interchange. How‐ ever, in riverine communities near urban centers, people have more accessibility to foreign

With regard to the way of life on the Amazon river, [18] indicated that these populations have an intense relationship with the cycles and dynamics of nature. Residents build their day-today activities and identities based on the river and forest surroundings, with a depth of knowledge about natural dynamics that reflects elaborate strategies for the use and manage‐

Knowledge is orally transferred from generation to generation. From a practical perspective, this is characteristic of such populations, who use symbols and rituals linked to hunting, fishing, and extractive activities. In addition to the practical sense, living with cyclical and

because of its historical lack of infrastructure investment [15].

goods and knowledge, thus incorporating values found in urban areas [17].

of Human Development)

ment of natural resources.

According to the author [5], more important than the use of such tools is the observation of daily activities and interactions in a given cultural context, which are full of meaning and express the concepts of parental care and how families are connected with the surrounding community [3, 9, 10]. In this sense, the studies by [1,2,5,3,7] are notable. In the case of Brazil, [11, 12, 13] contribute to the understanding of parenthood in different social groups.

The main interest in studying different cultural communities would, however, focus on the involvement of people in their communities, examining the nature and dynamics of generating individual lives and social practices of the community [3]. In other words, recognize what cultural practices familiar to every social group that permits examining and recognizing the organization, values, visions and social practices that are common and persistent in a given place, at a given social class or social group [3,4]

Considering that the roles performed by families are distinct in different cultural communities and in daily care practices, the objective of the present study was to describe the routines of two families who lived in Amazon river regions in the state of Pará, Brazil. These communities are inserted in the Amazonia forest, in which living activities are linked to nature with different degrees of isolation from urban centers.The figure 1 demonstrates this rich and distinctive ecological context.

In this perspective, the author [3] that made extensive transcultural research, tell us about the differences between the parental practices in terms of cultural prototypes, as being those which can valorize the autonomy within the caring practices, such as situation of the American families, and others that reforce the obedience and the sense of belonging to the group, such as the case of the rural communities. The same way, significant differences are found between the practices of the Minority World (typically represented by the white middle class from North America and Western Europe) and Majority (countries with low human development index) [8]. Such differences take in consideration aspects of the point of view economic and cultural and are evidenced from points such as: the attachment mother-baby, the shared responsibility, the probability of mortality, the size of the family, the roles played by the

Noteworthy is the fact that studies that compared educational practices between the Minori‐ tary World and poor rural communities in the Majoritary World led to a consensus that parental practices in those countries are a shortcoming [8,3,4].Therefore, parental practices in countries of the Minoritary World are considered more adequate, and such a trend is empha‐ sized by the use of measurement scales and tools for psychological investigation that advocate

An important topic to evidence is the fact that the studies interested in comparing the educa‐ tional practices observed between the Minority World and the impoverished rural commun‐ ities of the Majority World have conducted in a mistaken way to the consensus that the parental practices in those countries are presented as deficient [8, 3, 4]. In this sense, the parental practices present in the countries of the Minority World are taken as more adequate which trend is to reinforce by the use of measure scales and psychological investigation instruments

According to the author [5], more important than the use of such tools is the observation of daily activities and interactions in a given cultural context, which are full of meaning and express the concepts of parental care and how families are connected with the surrounding community [3, 9, 10]. In this sense, the studies by [1,2,5,3,7] are notable. In the case of Brazil,

The main interest in studying different cultural communities would, however, focus on the involvement of people in their communities, examining the nature and dynamics of generating individual lives and social practices of the community [3]. In other words, recognize what cultural practices familiar to every social group that permits examining and recognizing the organization, values, visions and social practices that are common and persistent in a given

Considering that the roles performed by families are distinct in different cultural communities and in daily care practices, the objective of the present study was to describe the routines of two families who lived in Amazon river regions in the state of Pará, Brazil. These communities are inserted in the Amazonia forest, in which living activities are linked to nature with different degrees of isolation from urban centers.The figure 1 demonstrates this rich and distinctive

[11, 12, 13] contribute to the understanding of parenthood in different social groups.

members of the family and the surrounding community [8, 3].

universal practices [5].

98 Parenting in South American and African Contexts

ecological context.

that profess them as universal practices [5].

place, at a given social class or social group [3,4]

**Figure 1.** The Amazonian context: the florest and housing typically riverine Amazon (collection of Laboratory Ecology of Human Development)

The Amazon river region consists of a forest rich in biodiversity, possessing 20% of the freshwater on the planet. Additionally, it represents 50% of the Brazilian territory and has approximately 11 million people, in which 62.4% live in the cities and 37.6% live in the country [14].The riverine area still has energetic resources and wealth and is rich with lush flora and fauna, but it is still considered an economically poor region compared with its natural richness because of its historical lack of infrastructure investment [15].

The riverine communities arranged in the Amazon forest are considered traditional, with occupations associated with waterways, cultures with riverine roots, and small-scale agricul‐ ture [16]. Although sharing a way of life and traditional cultures, significant differences are found among these communities in terms of access to public policies and economic develop‐ ment. Thus, riverine communities that live in geographic isolation have more financial stringency and depend more on forest resources and less on commercial interchange. How‐ ever, in riverine communities near urban centers, people have more accessibility to foreign goods and knowledge, thus incorporating values found in urban areas [17].

With regard to the way of life on the Amazon river, [18] indicated that these populations have an intense relationship with the cycles and dynamics of nature. Residents build their day-today activities and identities based on the river and forest surroundings, with a depth of knowledge about natural dynamics that reflects elaborate strategies for the use and manage‐ ment of natural resources.

Knowledge is orally transferred from generation to generation. From a practical perspective, this is characteristic of such populations, who use symbols and rituals linked to hunting, fishing, and extractive activities. In addition to the practical sense, living with cyclical and repetitive periods and guided by the dynamics of nature, distinct senses and meanings are generated in these populations, in which their routine tasks and social relationships develop more slowly [18].

The principal occupation of the men was extraction activities, followed by fishing. Women were housewives or artisans. The river and forest provided food and products for local

Parenting in Amazonian Riverside Context http://dx.doi.org/10.5772/57056 101

With regard to the availability of health services, no assistance was provided by the state, in addition to the lack of treated water, trash and sewer services, and complex health problems of the population (e.g., stomach and intestinal problems and malnutrition). Similar to the lack of health services, no investments were made in education. The only school in the community was located in the house of a local resident who provided only first-year elementary school

This community belongs to Combu Island, Belém county, Pará state. It has an approximate territorial size of 15 km² and is approximately 1.5 km from the capital Belém via waterway. It is characterized by floodplain forest with diversified flower composition in addition to

Considered the fourth largest island of Belém county, Combu Island is located at the margins of Guamá River. In this area, approximately 375 families or 1,700 residents live in and are clustered in four communities: Igarapé of the Combu, Igarapé of the Piriquitaquara, "Furo da Paciência," and the margin of Guamá River. The houses are predominantly made of wood and distributed along the margins of the waterways. The social and production structure is organized as a function of local natural resources, such as açaí and cacau harvesting, palm heart extraction, shrimping, fishing, and raising chickens, ducks, and pigs, which constituted

Access to Combu Island is via waterway on boats that leave daily from different Belém harbors. Such waterways play a strategic role in the transportation of goods and people. Work is performed with activities in the riverine area. Other services, including the marketing of plant and gathering products, health services, schools, shops, and amenities, occur in the urban

The houses were made of wood and covered with clay tiles or asbestos. They were built on stilts and located approximately 30 m from each other. Electricity was not available during data collection, but it became available in July 2011. Treated water was not yet available. Drinking water was obtained from a public tap in Belém and carried in bulk or plastic bottles

With regard to infrastructure, the community had a health center that was having difficulty with functioning because it was closed for some months due to a lack of physicians, equipment, and medicine. Aside from this health center, a municipal school was located in the igarapé itself, characterized by a typical Amazonia riverine style and made of wood on stilts. The school had two classrooms, a courtyard, a pantry, restrooms, a coordination room, and an external area available for recreation when the river level was low. In this community, almost all of the residents received benefits from the federal program "Bolsa Família" (family grant), a program

extraction activities, such as fish, shrimp, game animals, and açaí.

education.

**3.2. Piriquitaquara community**

the principal local activities.

context.

to the community.

enormous tourist and commercial attractions.

Based on interest in recognizing parenthood within the context such different development, the objective of the present study was to understand parental practices based on the descrip‐ tions of the routines of two riverine families and provide evidence of the relationships between cultural activities and the Amazonian context. We highlight the social activities during weekdays and weekends, with parenthood considered based on the daily activities that reflect these families' beliefs.