Once upon a Testis: The Tale of Cyclic Nucleotide Phosphodiesterase in Testicular Cancers

Abstract

Phosphodiesterases are key regulators that fine tune the intracellular levels of cyclic nucleotides, given their ability to hydrolyze cAMP and cGMP. They are critical regulators of cAMP/cGMP-mediated signaling pathways, modulating their downstream biological effects such as gene expression, cell proliferation, cell-cycle regulation but also inflammation and metabolic function. Recently, mutations in PDE genes have been identified and linked to human genetic diseases and PDEs have been demonstrated to play a potential role in predisposition to several tumors, especially in cAMP-sensitive tissues. This review summarizes the current knowledge and most relevant findings regarding the expression and regulation of PDE families in the testis focusing on PDEs role in testicular cancer development.

Full text

Citation: Campolo, F.; Assenza, M.R.;
Venneri, M.A.; Barbagallo, F. Once
upon a Testis: The Tale of Cyclic
Nucleotide Phosphodiesterase in
Testicular Cancers. Int. J. Mol. Sci.
2023,24, 7617. https://doi.org/
10.3390/ijms24087617
Academic Editor: Miroslav Chovanec
Received: 7 April 2023
Revised: 17 April 2023
Accepted: 19 April 2023
Published: 20 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
Once upon a Testis: The Tale of Cyclic Nucleotide
Phosphodiesterase in Testicular Cancers
Federica Campolo 1, Maria Rita Assenza 2, Mary Anna Venneri 1and Federica Barbagallo 2,*
1Department of Experimental Medicine, Sapienza University of Rome, 00161 Rome, Italy;
federica.campolo@uniroma1.it (F.C.); maryanna.venneri@uniroma1.it (M.A.V.)
2
Faculty of Medicine and Surgery, “Kore” University of Enna, 94100 Enna, Italy; mariarita.assenza@unikore.it
*Correspondence: federica.barbagallo@unikore.it
Abstract:
Phosphodiesterases are key regulators that fine tune the intracellular levels of cyclic
nucleotides, given their ability to hydrolyze cAMP and cGMP. They are critical regulators of
cAMP/cGMP-mediated signaling pathways, modulating their downstream biological effects such as
gene expression, cell proliferation, cell-cycle regulation but also inflammation and metabolic function.
Recently, mutations in PDE genes have been identified and linked to human genetic diseases and
PDEs have been demonstrated to play a potential role in predisposition to several tumors, especially
in cAMP-sensitive tissues. This review summarizes the current knowledge and most relevant find-
ings regarding the expression and regulation of PDE families in the testis focusing on PDEs role in
testicular cancer development.
Keywords: phosphodiesterases (PDEs); cAMP; cGMP; testis; testicular cancer
1. Introduction
Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP) are intracellular second messengers that play a central role in a plethora of sig-
naling pathways, involved in cell proliferation and differentiation, cell-cycle regulation,
Ca
2+
-dependent signaling, inflammation and metabolic function [
1
–
3
]. Intracellular levels
of cAMP and cGMP are finely regulated by adenylyl (AC) and guanylyl-cyclases, which
catalyze the synthesis of cAMP and cGMP from ATP and GTP, respectively. The increase
in intracellular cAMP and cGMP levels triggers the activation of several cellular effectors,
among which the mains are cAMP- and cGMP-activated protein kinases, PKA and PKG,
respectively [
4
,
5
]. The maintenance of cyclic nucleotide levels in physiological ranges is
dependent on the activity of phosphodiesterases (PDEs) that catalyze the hydrolysis of
cyclic nucleotides to the corresponding inactive non-cyclized monophosphate form (i.e.,
3
0
,5
0
-cGMP to 5
0
-GMP and 3
0
,5
0
-cAMP to 5
0
AMP, respectively) [
6
,
7
]. Mammalian PDEs
are classified into 11 families encoded by 21 different genes, that are grouped based on
their amino acid sequences, biochemical properties, affinities for cAMP and cGMP and
response to specific activators, inhibitors and effectors. Each PDE family consists of multi-
ple isoforms generated by alternative mRNA splicing or transcriptional processing, giving
rise to over 100 isoenzymes, which display different tissue expressions and intracellular
localization [
7
,
8
]. PDEs share a common structural organization, with a conserved carboxy-
terminal catalytic domain, while amino-terminal hydrophobic regulatory regions contain
structural determinants that target individual PDEs to different subcellular locations allow-
ing individual PDEs to specifically respond to different post-translational modifications and
signaling molecules [
9
–
11
]. Many of the PDE families contain amino-terminal subdomains,
such as GAF domains, which regulate the allosteric binding of cGMP to PDE2, PDE5, PDE6
and PDE11, or of cAMP to PDE10 [
12
]; upstream conserved regions, (e.g., in PDE4), harbors
a PKA consensus site [
13
]; Per-Arnt-Sim domains and receiver domains (e.g., in PDE8) [
14
].
Moreover, some PDE families contain phosphorylation sites, able to increase their enzyme
Int. J. Mol. Sci. 2023,24, 7617. https://doi.org/10.3390/ijms24087617 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 7617 2 of 26
activity, such as PDE5 phosphorylation site (Ser92) to PKG [
15
]; PDE1 presents two Calmod-
ulin (CaM)-binding domains activated by changes in intracellular [Ca
2+
] [
16
]; in PDE4, the
presence of extracellular signal-regulated kinase 2 plays a role in regulating their activity
and subcellular targeting [
17
]. The regulation of cyclic nucleotide signaling is thought to
be one of several pathways involved in tumor cells dissemination and function. In recent
years, we have witnessed a growing interest in the use of pharmacological inhibition of
PDEi as an anticancer strategy in several tumors [18].
In this review we first focused on the main findings of PDEs expression and the role
in the testis; then, we summarized the evidence of the aberrant expression of this class of
enzymes in testicular cancer.
Throughout this review, PDEs mRNA are capitalized and italicized, while the protein
products are capitalized but not italicized.
2. An Overview of Phosphodiesterase Families in Testis
2.1. PDE1
PDE1 is one of the first families to be identified and comprises Ca
2+
and calmodulin-
regulated PDEs displaying Ca
2+
/CaM binding domains. This family consists of three
subfamilies encoded by three different genes: PDE1A,PDE1B, and PDE1C (Figure 1).
Each PDE1 isoenzyme is present in tissues as splice variants that differ in molecular
weight, cellular and subcellular distribution and may play different roles accordingly. PDE1
enzymes are able to hydrolyze both cAMP and cGMP, with different affinities: PDE1A and
PDE1B exhibit higher affinity for cGMP whereas PDE1C possesses a similar affinity for
cAMP and cGMP.
Thus far, nine human and four mouse splice variants encoding for PDE1A have been
annotated, while two human and three mouse PDE1B1 splice variants are known [
19
]. Four
PDE1C splice variants have been reported in mouse, named PDE1C1–5 [
18
], while in human,
10 potential isoforms rising from an alternate promoter or alternate transcription start site
have been suggested; however, a consensus on their nomenclature is still missing [
20
,
21
].
PDE1A is widely expressed in various tissues, with some specific isoform restricted to the
brain [
21
]. Although several differences between mouse and human variants have been
highlighted it is commonly recognized that PDE1A10 (PDE1A9 in mice) is testis-specific
and its localization and function are conserved between the two species [
22
] (Tables 1and 2).
The elective tissue for PDE1B expression is the brain both in humans and mice with low or
undetectable levels in the testis regardless of the splice variants [
23
,
24
]. In humans, PDE1C
expression is high in both the heart and brain, while murine PDE1C1 and PDE1C5 isoforms
have been detected at high levels in the cerebellum [25].
The first evidence for the presence of calmodulin-dependent phosphodiesterases
within the testis emerged from biochemical studies by Purvis and collaborators in the
1980s [
26
,
27
]. They were able to isolate, by diethylaminoethyl cellulose chromatography,
three Ca
2+
-CaM dependent isoenzymes in immature rat testis, later on only the high-affinity
cGMP isoform was confirmed in both somatic and germ cell-enriched populations isolated
from 12-day-old rat testis [
28
,
29
] (Table 1). Some years later, the same group better clarified
that somatic cells possess a Ca
2+
-CaM-dependent high-affinity cGMP phosphodiesterase
whereas germ cells present a Ca
2+
-CaM-dependent high affinity for cGMP and a high and
low affinity for cAMP [
30
]. Despite the slight difference in the results, the two groups agreed
on the fact that CaM-PDE activities are developmentally regulated in rodent germ cells.

Int. J. Mol. Sci. 2023,24, 7617 3 of 26
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 27
Figure 1. Ca2+/CaM-stimulated PDE1 family. Human variants are depicted except for PDE1C (mouse
variants). This figure was gathered through analysis and cross-referencing of online databases
(hps://www.ensembl.org/ and hps://www.uniprot.org, accessed on 30 March 2023) for PDE1B
and PDE1C; PDE1A was adapted from [21]. Boxes and lines represent exons and introns, respec-
tively. The boxes with different colors indicate alternative exons. The maximum number of exons
illustrated is 17 and ellipsis indicate exons not shown.
The first evidence for the presence of calmodulin-dependent phosphodiesterases
within the testis emerged from biochemical studies by Purvis and collaborators in the
1980s [26,27]. They were able to isolate, by diethylaminoethyl cellulose chromatography,
three Ca2+-CaM dependent isoenzymes in immature rat testis, later on only the high-affin-
ity cGMP isoform was confirmed in both somatic and germ cell-enriched populations iso-
lated from 12-day-old rat testis [28,29] (Table 1). Some years later, the same group beer
clarified that somatic cells possess a Ca2+-CaM-dependent high-affinity cGMP phos-
phodiesterase whereas germ cells present a Ca2+-CaM-dependent high affinity for cGMP
and a high and low affinity for cAMP [30]. Despite the slight difference in the results, the
two groups agreed on the fact that CaM-PDE activities are developmentally regulated in
rodent germ cells.
Table 1. Expression of PDE family in rodent testis.
Gene Family Gene Cell Type References
PDE1
Un GCs [28]
PDE1A rSPT, eSPT, mSPT, SPZ [22,31,32]
PDE1C SPC, rSPT, eSPT, mSPT [32]
PDE2 PDE2A SPZ, Un [31,33]
PDE3 Un SPZ [31]
Figure 1.
Ca
2+
/CaM-stimulated PDE1 family. Human variants are depicted except for PDE1C
(mouse variants). This figure was gathered through analysis and cross-referencing of online databases
(https://www.ensembl.org/ and https://www.uniprot.org, accessed on 30 March 2023) for PDE1B
and PDE1C;PDE1A was adapted from [
21
]. Boxes and lines represent exons and introns, respectively.
The boxes with different colors indicate alternative exons. The maximum number of exons illustrated
is 17 and ellipsis indicate exons not shown.
Table 1. Expression of PDE family in rodent testis.
Gene Family Gene Cell Type References
PDE1
Un GCs [28]
PDE1A rSPT, eSPT, mSPT, SPZ [22,31,32]
PDE1C SPC, rSPT, eSPT, mSPT [32]
PDE2 PDE2A SPZ, Un [31,33]
PDE3
Un SPZ [31]
PDE3A SPC, Un [34]
PDE3B SPC [34]
PDE4
Un Un, SPZ [35,36]
PDE4A rSPT, pSPC [37–40]
PDE4B SCs, LCs, SPZ [31,35,41]
PDE4C pSPC, SPT, LCs, SPZ [31,38,39,41]
PDE4D
pSPCs, rSPT, eSPT, mSPT, SPZ, SCs
[31,40,42–45]
PDE5 PDE5A LCs, peritubular cells, Un [46–49]

Int. J. Mol. Sci. 2023,24, 7617 4 of 26
Table 1. Cont.
Gene Family Gene Cell Type References
PDE6
PDE6A SPZ, Un [31]
PDE6C LCs, SPZ [31,49]
PDE6D LCs, SPZ, Un [31,49]
PDE6G Un [31]
PDE6H Un [31]
PDE7 Un Un [50]
PDE7B pSPC [51]
PDE8 PDE8A LCs, pSPC, SPZ [31,41,49,52,53]
PDE8B Un, LCs SPZ [31,41,49]
PDE9 PDE9A Un, LCs [31,49]
PDE10 PDE10A SPZ, Un, LCs [31,49,54–56]
PDE11 PDE11A SPZ [31,57]
SPC: spermatocytes; pSPC: pachytene spermatocytes; SPT: spermatids; rSPT: round spermatids; eSPT: elongated
spermatids; SPZ: spermatozoa; GCs: germ cells; LCs: Leydig cells; SCs: Sertoli cells; UN: unspecified.
We need to wait 15 years until all PDE1 isoenzymes are cloned [
58
–
63
] before having
a better characterization of the stage and cell-specific expression of PDE1 enzymes in
murine testis [
32
]. A spatial and temporal expression pattern was observed for PDE1A
and PDE1C, with PDE1B considered absent. In particular, PDE1A mRNA was found in
round-to-elongated spermatids, while the protein expression was detected in the tails of
elongated and maturing spermatids but not in spermatocytes and spermatogonia [
22
,
32
].
PDE1C was expressed in the early-meiotic prophase through the meiotic and post-meiotic
stages [32].
In addition, a particulate CaM-PDE activity was noticed in the head and tailpieces of
rat caudal epididymal sperm [
15
]. These observations suggest that CaM-PDEs likely have
important roles in spermatogenesis and in the maturation of spermatozoa. Additionally,
capacitation was partially mediated by CaM-PDE activities [15].
In human spermatozoa PDE1 inhibitors selectively stimulated the acrosome reaction,
and given that PDE1A is the major form expressed in mature sperm; this variant was
attentional as an ideal candidate to play an important role in cyclic nucleotide regulation of
mature sperm function [22,36] (Table 2).
Table 2. Expression of PDE family in human testis.
Gene Family Gene Cell Type References
PDE1 PDE1A SPZ [36]
PDE2 PDE2A Un [64,65]
PDE3 PDE3A SPZ [66,67]
PDE4
PDE4A Un, SPZ [64–66]
PDE4B Un, SPZ [65,66]
PDE4C Un, SPZ [64,66]
PDE4D Un [65]
PDE5 PDE5A SPZ [66]
PDE6 PDE6B Un [64]
PDE7 PDE7B Un [64]

Int. J. Mol. Sci. 2023,24, 7617 5 of 26
Table 2. Cont.
Gene Family Gene Cell Type References
PDE8 PDE8A Un, LCs [64,65,68]
PDE8B Un, LCs [65]
PDE9 PDE9A Un [20,64]
PDE10 PDE10A Un [64]
PDE11 PDE11A SPC, SPT, LCs, Un [64,65,69]
SPC: spermatocytes; SPT: spermatids; SPZ: spermatozoa; LCs: Leydig cells; UN: unspecified.
2.2. PDE2
PDE2 hydrolyzes both cAMP and cGMP with higher affinity for the latest. It belongs
to the so-called “cGMP-stimulated PDE” since the binding of cGMP to the allosteric GAF-B
domain causes a conformational change that in turn stimulates cAMP hydrolysis. Given
this unique feature, PDE2A serves as a key regulator for the cAMP-cGMP crosstalk [70].
Three isoforms of PDE2 have been isolated so far: PDE2A1,PDE2A2 and PDE2A3
(Figure 2). These isoforms differ in their N-terminus which mediates their subcellular
localization [
71
]. PDE2A mRNA expression is similar in human and rodent tissue, in-
cluding the heart, liver, adrenal gland, platelets, brain, endothelial cells, neurons and
macrophages [70,72–76].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 27
PDE6 PDE6B Un [64]
PDE7 PDE7B Un [64]
PDE8 PDE8A Un, LCs [64,65,68]
PDE8B Un, LCs [65]
PDE9 PDE9A Un [20,64]
PDE10 PDE10A Un [64]
PDE11 PDE11A SPC, SPT, LCs, Un [64,65,69]
SPC: spermatocytes; SPT: spermatids; SPZ: spermatozoa; LCs: Leydig cells; UN: unspecified.
2.2. PDE2
PDE2 hydrolyzes both cAMP and cGMP with higher affinity for the latest. It belongs
to the so-called “cGMP-stimulated PDE” since the binding of cGMP to the allosteric GAF-
B domain causes a conformational change that in turn stimulates cAMP hydrolysis. Given
this unique feature, PDE2A serves as a key regulator for the cAMP-cGMP crosstalk [70].
Three isoforms of PDE2 have been isolated so far: PDE2A1, PDE2A2 and PDE2A3
(Figure 2). These isoforms differ in their N-terminus which mediates their subcellular lo-
calization [71]. PDE2A mRNA expression is similar in human and rodent tissue, including
the heart, liver, adrenal gland, platelets, brain, endothelial cells, neurons and macro-
phages [70,72–76].
Few reports characterized PDE2A expression in testis. Microarray data regarding the
expression of PDE genes in different human tissues reported a high level of PDE2A in the
human testis [64], later confirmed by the QuantiGene Bioplex Assay [65] (Table 2). Positive
results were obtained in human ejaculated spermatozoa [66] while low expression levels
were detected in murine spermatozoa [31] (Table 1). By Western blot an, undetectable sig-
nal was found in mouse testis extract, but there was mild to moderate staining by im-
munohistochemistry on rat, mouse and human testis slices (from lower to highest). In
particular, authors reported positive staining in subsets of spermatogenic and in Sertoli
cells, albeit this statement was not properly supported by images making it difficult to
discriminate to which cells the authors are referring to [33]. Even if its expression was
confirmed, the role of PDE2 in testis is still unknown.
Figure 2. cGMP-stimulated PDE2 family. Human variants are depicted. This figure was gathered
through analysis and cross-referencing of online databases (hps://www.ensembl.org/, and
hps://www.uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns,
respectively. The boxes with different colors indicate alternative exons. The maximum number of
exons illustrated is 17 and ellipsis indicate exons not shown.
2.3. PDE3
PDE3 hydrolyzes both cyclic nucleotides with higher affinity with cAMP. It has
earned the denomination of “cGMP-inhibited PDE” due to a distinctive feature: binding
of cGMP is able to inhibit cAMP hydrolysis.
Enzymes belonging to the PDE3 family are transcribed from two different genes,
PDE3A and PDE3B [77,78]. For PDE3A, three variants have been described, differing only
in the lengths of their N-terminal sequences [10,79] and not in their basal catalytic activity
[80]. Up to now, PDE3B is the only isoform annotated [77] and possesses similar catalytic
activity to PDE3A (Figure 3) [81]. PDE3A is expressed in the heart, vascular smooth
Figure 2.
cGMP-stimulated PDE2 family. Human variants are depicted. This figure was gathered
through analysis and cross-referencing of online databases (https://www.ensembl.org/, and https:
//www.uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns,
respectively. The boxes with different colors indicate alternative exons. The maximum number of
exons illustrated is 17 and ellipsis indicate exons not shown.
Few reports characterized PDE2A expression in testis. Microarray data regarding the
expression of PDE genes in different human tissues reported a high level of PDE2A in the
human testis [
64
], later confirmed by the QuantiGene Bioplex Assay [
65
] (Table 2). Positive
results were obtained in human ejaculated spermatozoa [
66
] while low expression levels
were detected in murine spermatozoa [
31
] (Table 1). By Western blot an, undetectable
signal was found in mouse testis extract, but there was mild to moderate staining by
immunohistochemistry on rat, mouse and human testis slices (from lower to highest). In
particular, authors reported positive staining in subsets of spermatogenic and in Sertoli
cells, albeit this statement was not properly supported by images making it difficult to
discriminate to which cells the authors are referring to [
33
]. Even if its expression was
confirmed, the role of PDE2 in testis is still unknown.
2.3. PDE3
PDE3 hydrolyzes both cyclic nucleotides with higher affinity with cAMP. It has earned
the denomination of “cGMP-inhibited PDE” due to a distinctive feature: binding of cGMP
is able to inhibit cAMP hydrolysis.
Enzymes belonging to the PDE3 family are transcribed from two different genes,
PDE3A and PDE3B [
77
,
78
]. For PDE3A, three variants have been described, differing
only in the lengths of their N-terminal sequences [
10
,
79
] and not in their basal catalytic
activity [
80
]. Up to now, PDE3B is the only isoform annotated [
77
] and possesses similar

Int. J. Mol. Sci. 2023,24, 7617 6 of 26
catalytic activity to PDE3A (Figure 3) [
81
]. PDE3A is expressed in the heart, vascular
smooth muscle regulating myocardial and smooth muscle contractility, platelets, oocyte
and kidney whereas PDE3B is enriched in vascular smooth muscle, adipocytes, hepatocytes,
kidney, b cells, T lymphocytes and macrophages and it is involved in hormonal regulation
of lipolysis and glycogenolysis [82,83].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 27
muscle regulating myocardial and smooth muscle contractility, platelets, oocyte and kid-
ney whereas PDE3B is enriched in vascular smooth muscle, adipocytes, hepatocytes, kid-
ney, b cells, T lymphocytes and macrophages and it is involved in hormonal regulation of
lipolysis and glycogenolysis [82,83].
The first evidence of PDE3 transcripts in testis arises from the results obtained on rat
testis where PDE3A was detected in vessels and PDE3B was found in primary spermato-
cytes [34] (Table 1). No other characterization of the cellular localization of these two en-
zymes was carried out, leaving it unclear if the transcripts are also translated into func-
tional proteins in these subsets of cells. Enzyme activity, immunocytochemical localiza-
tion and immunobloing for PDE3A were applied on human spermatozoa raveling that
it is expressed and localized on the post-acrosomal segment of the sperm head [66,67]
(Table 2); however, its inhibition by milrinone did not significantly stimulate capacitation
or hyperactivation suggesting that PDE3 does not have a major role in sperm function
[67].
Figure 3. cGMP-inhibited PDE gene family. Human variants are depicted. This figure was gathered
through analysis and cross-referencing of online databases (hps://www.ensembl.org/ and
hps://www.uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns,
respectively. The boxes with different colors indicate alternative exons. The maximum number of
exons illustrated is 17 and ellipsis indicate exons not shown.
2.4. PDE4
PDE4 enzymes constitute the majority of cAMP-selective PDEs. Four distinct genes
(PDE4A, PDE4B, PDE4C and PDE4D) encode the PDE4 family of enzymes, each of these
genes produce a plethora of transcript variants and different protein isoforms (Figure 4).
PDE4 subtypes and isoforms possess tissue- and cell type-specific expression but also they
can have intracellular compartmentalization specificity. PDE4 enzyme expression is ubiq-
uitous with variant-specific tissue distribution [82].
In 1992, Contis group performed a Northen blot assay, using a probe specific for
PDE4A, to analyze its expression on isolated spermatogenic cells. They detected a 4.0-kb
PDE4 mRNA in mouse and rat pachytene spermatocytes and five transcripts mRNAs in
round spermatids, while a lower amount of transcripts was found in condensing spermat-
ocytes/residual bodies [37] (Table 1). Similar results were obtained by an in situ hybridi-
zation approach performed by Morena and colleagues. They detected a high PDE4A sig-
nal in round spermatids, later aributed to PDE4A7 isoform [38], that declined in elongat-
ing spermatids [39,40]. Western bloing using PDE4 subtype-selective antibodies con-
firmed the paern of mRNA expression studies conducted by in situ hybridization [35].
Developmental studies on rodents are also consistent with the presence of PDE4A8
mRNA and expression of PDE4A8 and 88-kDa PDE4A protein at 20–30 days of age.
PDE4B mRNAs and protein were found primarily in the Sertoli and Leydig cells [35],
whereas PDE4C maximal expression was detected in stages VIII-XIII of the seminiferous
epithelium indicating that it is expressed preferentially in middle–late pachytene sper-
matocytes [39]. As for PDE4B also PDE4D, in particular PDE4D1 and PDE4D2, are ex-
pressed in immature Sertoli cells and regulated by a follicle-stimulating hormone (FSH)-
Figure 3.
cGMP-inhibited PDE gene family. Human variants are depicted. This figure was gathered
through analysis and cross-referencing of online databases (https://www.ensembl.org/ and https:
//www.uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns,
respectively. The boxes with different colors indicate alternative exons. The maximum number of
exons illustrated is 17 and ellipsis indicate exons not shown.
The first evidence of PDE3 transcripts in testis arises from the results obtained on
rat testis where PDE3A was detected in vessels and PDE3B was found in primary sper-
matocytes [
34
] (Table 1). No other characterization of the cellular localization of these
two enzymes was carried out, leaving it unclear if the transcripts are also translated into
functional proteins in these subsets of cells. Enzyme activity, immunocytochemical local-
ization and immunoblotting for PDE3A were applied on human spermatozoa raveling
that it is expressed and localized on the post-acrosomal segment of the sperm head [
66
,
67
]
(Table 2); however, its inhibition by milrinone did not significantly stimulate capacitation
or hyperactivation suggesting that PDE3 does not have a major role in sperm function [
67
].
2.4. PDE4
PDE4 enzymes constitute the majority of cAMP-selective PDEs. Four distinct genes
(PDE4A,PDE4B,PDE4C and PDE4D) encode the PDE4 family of enzymes, each of these
genes produce a plethora of transcript variants and different protein isoforms (Figure 4).
PDE4 subtypes and isoforms possess tissue- and cell type-specific expression but also
they can have intracellular compartmentalization specificity. PDE4 enzyme expression is
ubiquitous with variant-specific tissue distribution [82].
In 1992, Conti’s group performed a Northen blot assay, using a probe specific for
PDE4A, to analyze its expression on isolated spermatogenic cells. They detected a 4.0-kb
PDE4 mRNA in mouse and rat pachytene spermatocytes and five transcripts mRNAs in
round spermatids, while a lower amount of transcripts was found in condensing spermato-
cytes/residual bodies [
37
] (Table 1). Similar results were obtained by an in situ hybridiza-
tion approach performed by Morena and colleagues. They detected a high PDE4A signal
in round spermatids, later attributed to PDE4A7 isoform [
38
], that declined in elongating
spermatids [
39
,
40
]. Western blotting using PDE4 subtype-selective antibodies confirmed
the pattern of mRNA expression studies conducted by in situ hybridization [
35
]. Devel-
opmental studies on rodents are also consistent with the presence of PDE4A8 mRNA and
expression of PDE4A8 and 88-kDa PDE4A protein at 20–30 days of age. PDE4B mRNAs and
protein were found primarily in the Sertoli and Leydig cells [
35
], whereas PDE4C maximal
expression was detected in stages VIII-XIII of the seminiferous epithelium indicating that it
is expressed preferentially in middle–late pachytene spermatocytes [
39
]. As for PDE4B also
PDE4D, in particular PDE4D1 and PDE4D2, are expressed in immature Sertoli cells and
regulated by a follicle-stimulating hormone (FSH)-cAMP-mediated mechanism [
42
–
44
].

Int. J. Mol. Sci. 2023,24, 7617 7 of 26
Given the different electrophoretic mobilities, five immunoreactive species were detected
in immature Sertoli cells [45].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 27
cAMP-mediated mechanism [42–44]. Given the different electrophoretic mobilities, five
immunoreactive species were detected in immature Sertoli cells [45].
Figure 4. PDE4 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [84]).
The longest PDE4D transcripts were also enriched in pachytene spermatocyte and
round spermatids [40]. Translated protein appears only in a region surrounding the
Figure 4.
PDE4 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown (adapted from [84]).

Int. J. Mol. Sci. 2023,24, 7617 8 of 26
The longest PDE4D transcripts were also enriched in pachytene spermatocyte and
round spermatids [
40
]. Translated protein appears only in a region surrounding the
acrosome of elongating and maturing spermatids in close proximity to microtubules present
in the transitory structure of the manchette indicating that PDE4D mRNA is not efficiently
translated at round spermatids stages but it reaches a maximal intensity at steps 18–19
before spermiation [
40
]. PDE4A, PDE4B and PDE4D have been detected with a similar rate
of expression in whole human testis extract [65] (Table 2).
In mouse spermatozoa, immunolocalization of PDE4 revealed an intense signal for
isoform D in the principal piece and across the entire acrosome and for 4A in the flagellum,
while 4B and 4C were considered expressed at very low levels or absent [
31
]. PDE4A,4B,
4C but not 4D transcripts were detected in human spermatozoa [
66
] and their inhibition
enhanced sperm motility, phosphorylation of membrane proteins [
85
] without affecting
acrosome reaction [
36
]. Surprisingly, a proteomic study revealed only PDE4D but no other
PDE4 isoforms expression in human sperm [86].
MA10 mouse Leydig cell line and primary rodent Leydig cells expressing PDE4B
and 4C and PDE4 inhibition by rolipram are able to regulate steroid synthesis under basal
conditions and upon luteinizing hormone (LH) stimulation [
41
]. PDE4 and PDE8A (see
forehead), coordinate steroidogenesis through several layers of control such as transcription,
lipid and glucose metabolism, endocytosis and vesicle transport to facilitate maximal
steroid output and to assure timely and adequate testosterone secretion in response to
LH [87].
2.5. PDE5
PDE5A is characterized by high specificity for cGMP [
88
,
89
]. Three murine and human
isoforms have been characterized so far that are transcribed from different promoters,
producing three N-terminal variants (PDE5A1,PDE5A2,PDE5A3) [
47
,
90
,
91
] (Figure 5).
PDE5A transcripts are widely expressed but high levels have been detected in several
sections of the digestive system, lung, platelets, cerebellum, kidney, vascular smooth
muscle cells, skeletal and cardiac muscle [
92
–
98
], and several endocrine glands, including
testis [47,65,99].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 27
acrosome of elongating and maturing spermatids in close proximity to microtubules pre-
sent in the transitory structure of the manchee indicating that PDE4D mRNA is not effi-
ciently translated at round spermatids stages but it reaches a maximal intensity at steps
18–19 before spermiation [40]. PDE4A, PDE4B and PDE4D have been detected with a sim-
ilar rate of expression in whole human testis extract [65] (Table 2).
In mouse spermatozoa, immunolocalization of PDE4 revealed an intense signal for
isoform D in the principal piece and across the entire acrosome and for 4A in the flagel-
lum, while 4B and 4C were considered expressed at very low levels or absent [31]. PDE4A,
4B, 4C but not 4D transcripts were detected in human spermatozoa [66] and their inhibi-
tion enhanced sperm motility, phosphorylation of membrane proteins [85] without affect-
ing acrosome reaction [36]. Surprisingly, a proteomic study revealed only PDE4D but no
other PDE4 isoforms expression in human sperm [86].
MA10 mouse Leydig cell line and primary rodent Leydig cells expressing PDE4B and
4C and PDE4 inhibition by rolipram are able to regulate steroid synthesis under basal
conditions and upon luteinizing hormone (LH) stimulation [41]. PDE4 and PDE8A (see
forehead), coordinate steroidogenesis through several layers of control such as transcrip-
tion, lipid and glucose metabolism, endocytosis and vesicle transport to facilitate maximal
steroid output and to assure timely and adequate testosterone secretion in response to LH
[87].
2.5. PDE5
PDE5A is characterized by high specificity for cGMP [88,89]. Three murine and hu-
man isoforms have been characterized so far that are transcribed from different promot-
ers, producing three N-terminal variants (PDE5A1, PDE5A2, PDE5A3) [47,90,91] (Figure
5). PDE5A transcripts are widely expressed but high levels have been detected in several
sections of the digestive system, lung, platelets, cerebellum, kidney, vascular smooth mus-
cle cells, skeletal and cardiac muscle [92–98], and several endocrine glands, including tes-
tis [47,65,99].
Immunolocalization of PDE5A in prepuberal and adult testis is restricted to Leydig
and peritubular cells, proposing cGMP-mediated processes to influence not only the ves-
sel dilatation, but also the testosterone synthesis by Leydig cells [46].
Figure 5. PDE5 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [93]).
This idea was later confirmed in mice chronically treated with sildenafil. The treat-
ment induced several changes in Leydig cells such as vesicular smooth endoplasmic re-
ticulum, large vacuoles scaered through the cytoplasm and enlarged mitochondria, hall-
marks of an activated steroid-secreting cell [48]. The fact that sildenafil-treated mice pre-
sented also increased levels of total testosteron e sugge st tha t PDE5/PKG c ould be involved
in the modulation of androgen biosynthesis [48,49,100]. The effect of PDE5i on the human
chorionic gonadotropin/LH-induced steroidogenic pathway was also investigated in
HEK293 and MLTC-1 cell lines by Forster resonance energy transfer-based biosensors re-
vealing that PDE5i was able to enhance the conversion of progesterone-to-testosterone in
Figure 5.
PDE5 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown (adapted from [93]).
Immunolocalization of PDE5A in prepuberal and adult testis is restricted to Leydig
and peritubular cells, proposing cGMP-mediated processes to influence not only the vessel
dilatation, but also the testosterone synthesis by Leydig cells [46].
This idea was later confirmed in mice chronically treated with sildenafil. The treatment
induced several changes in Leydig cells such as vesicular smooth endoplasmic reticulum,
large vacuoles scattered through the cytoplasm and enlarged mitochondria, hallmarks
of an activated steroid-secreting cell [
48
]. The fact that sildenafil-treated mice presented
also increased levels of total testosterone suggest that PDE5/PKG could be involved
in the modulation of androgen biosynthesis [
48
,
49
,
100
]. The effect of PDE5i on the hu-
man chorionic gonadotropin/LH-induced steroidogenic pathway was also investigated
in HEK293 and MLTC-1 cell lines by Forster resonance energy transfer-based biosensors

Int. J. Mol. Sci. 2023,24, 7617 9 of 26
revealing that PDE5i was able to enhance the conversion of progesterone-to-testosterone in
a cAMP-independent manner [
101
]. This effect was later explained by a cross-interaction
between PDE5i and cAMP-specific PDE8A/PDE8B leading to an increase in cAMP and sex
hormones levels [
101
,
102
]. Interestingly, recent results indicate that long-term sildenafil
treatment improves testicular steroidogenesis as well as the sensitivity of Leydig cells
to gonadotropic stimulation and ameliorate the atrophy of seminiferous tubules during
aging [103].
Although Sertoli cells seem not to express PDE5A [
46
], some studies demonstrated
that PDE5A regulates Sertoli cell secretion. In Azoospermic men, vardenafil modulates
Sertoli cell secretory function and results in androgen-binding protein enhancement, a
biological marker of Sertoli cell secretion [
104
] may be due an indirect positive effect on
peritubular cells or on the secretory function of Leydig cells. The literature regarding
PDE5A localization in human testis is lacking.
Given the high specificity and safety of PDE5 inhibitors, several clinical trials have
been conducted. In two clinical trials performed on healthy volunteers, sildenafil does
not modify seminal parameters and acrosome reaction [
105
,
106
]; on the other hand, either
sildenafil citrate or 8-Bromo-cGMP treatments increased sperm-zona pellucida binding,
suggesting that PDE5i can be used to enhance sperm motility and oocyte binding [
106
].
Jannini et al. investigated the effect of orally administered sildenafil in healthy men. In
this study, no effect of sildenafil administration was observed regarding sperm motility,
concentration, or in the total number of ejaculated spermatozoa. However, when silde-
nafil was administrated before the second postcoital test, it increase sperm number and
motility [
107
]. Several attempts to clarify the role of PDE5A by inhibiting its activity have
been performed
in vitro
on human spermatozoa, where its expression was confirmed by
Richter and colleagues [
66
] (Table 2). The effects of sildenafil, vardenafil and tadalafil on
the motility, viability, membrane integrity and functional capacity of human spermatozoa
are controversial [
108
–
111
]. Some authors reported positive effects of sildenafil on sperm
viability, sperm motility, sperm forward progression and acrosome reaction attributing
these effects to cGMP-regulated calcium influx [
112
]. Others obtained no significant effects
leaving the debate still open.
2.6. PDE6
Photoreceptor cell-specific PDE6 protein complex comprises three genes: PDE6A,
PDE6B and PDE6C encoding the catalytic subunits; PDE6G and PDE6H genes encoding
the inhibitory subunits and PDE6D responsible for their solubilization (Figure 6) [113].
PDE6 is the primary regulator of cytoplasmic cGMP concentration in rod and cone
photoreceptors being almost exclusively expressed in the mammalian retina and in the
pineal gland [
114
]. PDE6 expression in rodent testis has been first demonstrated by An-
dric et al. They found that chronic treatment with sildenafil of rat Leydig cells reduces
PDE6C without affecting PDE6D mRNA expression [
49
] (Table 1). Moreover, Baxendale
and Fraser investigated the presence and function of PDEs in mouse testis and mature
spermatozoa demonstrating that PDE6A and PDE6D transcripts are expressed in both,
PDE6G and PDE6H are expressed in testis only while PDE6C expression is restricted to
mature spermatozoa [31].
Microarray analysis of phosphodiesterases expression on human tissues revealed that
PDE6A and PDE6C are undetectable while PDE6B is expressed at moderate levels in normal
testis [64] (Table 2).

Int. J. Mol. Sci. 2023,24, 7617 10 of 26
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 27
Figure 6. PDE6 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown.
PDE6 is the primary regulator of cytoplasmic cGMP concentration in rod and cone
photoreceptors being almost exclusively expressed in the mammalian retina and in the
pineal gland [114]. PDE6 expression in rodent testis has been first demonstrated by Andric
et al. They found that chronic treatment with sildenafil of rat Leydig cells reduces PDE6C
without affecting PDE6D mRNA expression [49] (Table 1). Moreover, Baxendale and Fra-
ser investigated the presence and function of PDEs in mouse testis and mature spermato-
zoa demonstrating that PDE6A and PDE6D transcripts are expressed in both, PDE6G and
PDE6H are expressed in testis only while PDE6C expression is restricted to mature sper-
matozoa [31].
Microarray analysis of phosphodiesterases expression on human tissues revealed
that PDE6A and PDE6C are undetectable while PDE6B is expressed at moderate levels in
normal testis [64] (Table 2).
2.7. PDE7
PDE7 is a cAMP-selective phosphodiesterase encoded by two genes PDE7A and
PDE7B that, following alternative splicing, give rise to three PDE7A isoforms (PDE7A1,
PDE7A2, and PDE7A3); for PDE7B it is widely recognized only one translated isoform
[115] (Figure 7).
Figure 6.
PDE6 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown.
2.7. PDE7
PDE7 is a cAMP-selective phosphodiesterase encoded by two genes PDE7A and
PDE7B that, following alternative splicing, give rise to three PDE7A isoforms (PDE7A1,
PDE7A2, and PDE7A3); for PDE7B it is widely recognized only one translated isoform [
115
]
(Figure 7).
Timothy Bloom and Joseph Beavo first reported a barely detectable PDE7 expression
signal in mouse testis performing a ribonuclease protection analysis [
50
] (Table 1), also
revealing high expression levels of PDE7 in mouse skeletal muscle. In human tissues, the
highest expression levels are detectable in T lymphocytes [
116
]. Some years later, Sasaki
and coworkers characterized PDE7 expression in rat testis. Using northern and in situ
hybridization analyses, they showed that PDE7B transcripts were particularly abundant
in rat spermatocytes [
51
]. The expression of PDE7 in the human testis is still debated.
The only data come from a microarray analysis of PDE expression on human tissues that
showed that PDE7B is expressed at moderate levels in normal testis while PDE7A levels
are undetectable [64] (Table 2).

Int. J. Mol. Sci. 2023,24, 7617 11 of 26
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 11 of 27
Figure 7. PDE7 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown.
Timothy Bloom and Joseph Beavo first reported a barely detectable PDE7 expression
signal in mouse testis performing a ribonuclease protection analysis [50] (Table 1), also
revealing high expression levels of PDE7 in mouse skeletal muscle. In human tissues, the
highest expression levels are detectable in T lymphocytes [116]. Some years later, Sasaki
and coworkers characterized PDE7 expression in rat testis. Using northern and in situ
hybridization analyses, they showed that PDE7B transcripts were particularly abundant
in rat spermatocytes [51]. The expression of PDE7 in the human testis is still debated. The
only data come from a microarray analysis of PDE expression on human tissues that
showed that PDE7B is expressed at moderate levels in normal testis while PDE7A levels
are undetectable [64] (Table 2).
2.8. PDE8
PDE8 is a highly selective cAMP hydrolyzing enzyme and consists of two genes,
PDE8A and PDE8B that due to alternative splicing processes, generate five splice variants:
PDE8A1–PDE8A5 and PDE8B1–PDE8B5 [117,118] (Figure 8). Both PDE8 genes are widely
expressed in all steroidogenic cell types with PDE8A mostly expressed in the testis and T-
cells and PDE8B mainly distributed in the brain and thyroid gland making PDE8 a key
player in T-cell activation, thyroid hormones production, sperm and Leydig cell functions
and cardiac functions [119,120].
The expression and the role of PDE8 in mouse testis were deeply analyzed by Vasta
et al. that, using PDE8A knockout mice, provided evidence of PDE8A pivotal role in
steroidogenesis [52] (Table 1). Some years later, Shimizu-Albergine et al. added important
details to this issue demonstrating that PDE8A and PDE8B work in concert to regulate
steroid production [41]. This was further supported by the finding that, although PDE8A
and PDE8B hydrolyze distinct cAMP pools to regulate basal rates of steroidogenesis, max-
imal steroid production requires the inhibition of both isoforms [121,122]. The functional
involvement of PDE8 in the regulation of mouse steroidogenesis was also supported by a
phosphoproteomics analysis on the steroidogenic MA10 cell model stimulated with selec-
tive PDE8i. This analysis, tracing global phosphoproteome dynamics in response to
cAMP/PKA activation, clearly demonstrated a specific role of PDE8 in the regulation of
steroidogenic gene transcription [87].
Figure 7.
PDE7 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown.
2.8. PDE8
PDE8 is a highly selective cAMP hydrolyzing enzyme and consists of two genes,
PDE8A and PDE8B that due to alternative splicing processes, generate five splice variants:
PDE8A1–PDE8A5 and PDE8B1–PDE8B5 [
117
,
118
] (Figure 8). Both PDE8 genes are widely
expressed in all steroidogenic cell types with PDE8A mostly expressed in the testis and
T-cells and PDE8B mainly distributed in the brain and thyroid gland making PDE8 a key
player in T-cell activation, thyroid hormones production, sperm and Leydig cell functions
and cardiac functions [119,120].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 12 of 27
Figure 8. PDE8 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [67]).
Several studies investigated the role of PDE8 in the whole testis. In one of those stud-
ies, PDE8A mRNA was specifically found in pachytene spermatocytes, suggesting a po-
tential role in germ cell development [53].
The expression of PDE8 has also been immunodetected in mouse spermatozoa by
different groups [41]. To this regard, Baxendale and Fraser, investigating the presence and
function of PDEs in mouse testis and mature spermatozoa, demonstrated that PDE8A and
PDE8B transcripts are both expressed in mature testis while only PDE8A is expressed in
mature spermatozoa [31]. PDE8 expression in rodent testis has been further demonstrated
by Andric et al. that analyze PDEs expression following chronic treatment with sildenafil
demonstrating that rat Leydig cells express PDE8A and PDE8B mRNAs and sildenafil is
not able to modify their levels [49].
Although there are many demonstrations on rodents, PDE8 expression in human testis
has been poorly investigated due to the known limitations related to the availability of hu-
man specimens. A demonstration of the presence of PDE8 in human testis comes from stud-
ies conducted by Wang et al. that, analyzing the tissue distribution of human PDE8A
isoforms, found that PDE8A1 transcript is mos t abundant in testis while PDE8A2 expression
levels are highest in spleen followed by testis [68]. Moreover, a microarray analysis of phos-
phodiesterases expression on human tissues showed that PDE8A is expressed at moderate
levels in normal testis while PDE8B levels are undetectable. We recently demonstrated that
human Leydig cells express both PDE8A and PDE8B isoforms and that PDE8A is also highly
expressed in specific spermatogenic stages, suggesting a potentially pivotal role of PDE8A
in controlling key events of maturation of human sperm [65] (Table 2).
2.9. PDE9
PDE9 is the cGMP-hydrolyzing PDE with the highest affinity for cGMP among all
PDE families and is encoded by a PDE9A gene that, following alternative splicing, gives
rise to five encoding transcripts (PDE9A1–PDE9A6, the latest originally named PDE9A5)
[123] (Figure 9). Human PDE9A shows the highest expression levels in the spleen and
brain particularly in Purkinje neurons and cerebellum [124,125].
Figure 8.
PDE8 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown (adapted from [67]).
The expression and the role of PDE8 in mouse testis were deeply analyzed by Vasta
et al. that, using PDE8A knockout mice, provided evidence of PDE8A pivotal role in
steroidogenesis [
52
] (Table 1). Some years later, Shimizu-Albergine et al. added important
details to this issue demonstrating that PDE8A and PDE8B work in concert to regulate
steroid production [
41
]. This was further supported by the finding that, although PDE8A
and PDE8B hydrolyze distinct cAMP pools to regulate basal rates of steroidogenesis, maxi-

Int. J. Mol. Sci. 2023,24, 7617 12 of 26
mal steroid production requires the inhibition of both isoforms [
121
,
122
]. The functional
involvement of PDE8 in the regulation of mouse steroidogenesis was also supported by
a phosphoproteomics analysis on the steroidogenic MA10 cell model stimulated with
selective PDE8i. This analysis, tracing global phosphoproteome dynamics in response to
cAMP/PKA activation, clearly demonstrated a specific role of PDE8 in the regulation of
steroidogenic gene transcription [87].
Several studies investigated the role of PDE8 in the whole testis. In one of those
studies, PDE8A mRNA was specifically found in pachytene spermatocytes, suggesting a
potential role in germ cell development [53].
The expression of PDE8 has also been immunodetected in mouse spermatozoa by
different groups [
41
]. To this regard, Baxendale and Fraser, investigating the presence and
function of PDEs in mouse testis and mature spermatozoa, demonstrated that PDE8A and
PDE8B transcripts are both expressed in mature testis while only PDE8A is expressed in
mature spermatozoa [
31
]. PDE8 expression in rodent testis has been further demonstrated
by Andric et al. that analyze PDEs expression following chronic treatment with sildenafil
demonstrating that rat Leydig cells express PDE8A and PDE8B mRNAs and sildenafil is
not able to modify their levels [49].
Although there are many demonstrations on rodents, PDE8 expression in human testis
has been poorly investigated due to the known limitations related to the availability of
human specimens. A demonstration of the presence of PDE8 in human testis comes from
studies conducted by Wang et al. that, analyzing the tissue distribution of human PDE8A
isoforms, found that PDE8A1 transcript is most abundant in testis while PDE8A2 expres-
sion levels are highest in spleen followed by testis [
68
]. Moreover, a microarray analysis
of phosphodiesterases expression on human tissues showed that PDE8A is expressed at
moderate levels in normal testis while PDE8B levels are undetectable. We recently demon-
strated that human Leydig cells express both PDE8A and PDE8B isoforms and that PDE8A
is also highly expressed in specific spermatogenic stages, suggesting a potentially pivotal
role of PDE8A in controlling key events of maturation of human sperm [65] (Table 2).
2.9. PDE9
PDE9 is the cGMP-hydrolyzing PDE with the highest affinity for cGMP among all PDE
families and is encoded by a PDE9A gene that, following alternative splicing, gives rise to
five encoding transcripts (PDE9A1–PDE9A6, the latest originally named PDE9A5) [
123
]
(Figure 9). Human PDE9A shows the highest expression levels in the spleen and brain
particularly in Purkinje neurons and cerebellum [124,125].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 13 of 27
Figure 9. PDE9 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [122]).
PDE9A protein expression is highly conserved among species being widely distrib-
uted throughout mouse and rat brains with different regional expressions [126,127]. Stud-
ies on PDE9A knockout mice revealed that cGMP levels were increased in the brain cortex,
hippocampus striatum, cerebellum and cerebrospinal fluid and the chronic treatment of
wild-type mice with a PDE9 selective inhibitor (PF-4181366) increased cGMP levels in the
same brain regions as well as in the cerebrospinal fluid [128]. The analysis of PDEs expres-
sion in rodent testis revealed that PDE9A mRNA is detectable in mouse testis [31] and in
Leydig cells obtained from rats [49] while expression data on human testis are controver-
sial [20,64] (Tables 1 and 2).
2.10. PDE10
PDE10 is a dual cAMP/cGMP hydrolyzing enzyme encoded by a single gene,
PDE10A present in two major variants, PDE10A1 and PDE10A2 [54,55] (Figure 10). It
shows a higher affinity for cAMP and may function in vivo as a cAMP-inhibited cGMP
PDE [129].
Figure 10. PDE10 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [130]).
PDE10A is mainly expressed in the thyroid, pituitary glands and brain; it has been
suggested as a regulator of learning and memory processes [131].
The expression of PDE10A has been investigated by Baxendale and Fraser, analyzing
the presence and function of murine PDEs and demonstrating that PDE10A transcripts
are expressed in testis but not in mature spermatozoa [31] (Table 1). Other works con-
firmed this expression paern in rodent testis with different technical approaches [54].
Among them, Andric et al. clearly demonstrated that rat Leydig cells express PDE10 tran-
script and chronic treatment with a selective Pde5i is not able to modify PDE10 mRNA
expression levels [49]. A decade before, Fujishige et al. reported a strong PDE10A im-
munoblot signal corresponding to high enzymatic activity in rat testis and striatum [132],
removing any reasonable doubt on the presence of PDE10 in these two organs.
Figure 9.
PDE9 gene family. Human variants are depicted. This figure was gathered through analysis
and cross-referencing of online databases (https://www.ensembl.org/ and https://www.uniprot.org,
accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The boxes
with different colors indicate alternative exons. The maximum number of exons illustrated is 17 and
ellipsis indicate exons not shown (adapted from [122]).

Int. J. Mol. Sci. 2023,24, 7617 13 of 26
PDE9A protein expression is highly conserved among species being widely distributed
throughout mouse and rat brains with different regional expressions [
126
,
127
]. Studies
on PDE9A knockout mice revealed that cGMP levels were increased in the brain cortex,
hippocampus striatum, cerebellum and cerebrospinal fluid and the chronic treatment of
wild-type mice with a PDE9 selective inhibitor (PF-4181366) increased cGMP levels in
the same brain regions as well as in the cerebrospinal fluid [
128
]. The analysis of PDEs
expression in rodent testis revealed that PDE9A mRNA is detectable in mouse testis [
31
]
and in Leydig cells obtained from rats [
49
] while expression data on human testis are
controversial [20,64] (Tables 1and 2).
2.10. PDE10
PDE10 is a dual cAMP/cGMP hydrolyzing enzyme encoded by a single gene, PDE10A
present in two major variants, PDE10A1 and PDE10A2 [
54
,
55
] (Figure 10). It shows a higher
affinity for cAMP and may function in vivo as a cAMP-inhibited cGMP PDE [129].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 13 of 27
Figure 9. PDE9 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [122]).
PDE9A protein expression is highly conserved among species being widely distrib-
uted throughout mouse and rat brains with different regional expressions [126,127]. Stud-
ies on PDE9A knockout mice revealed that cGMP levels were increased in the brain cortex,
hippocampus striatum, cerebellum and cerebrospinal fluid and the chronic treatment of
wild-type mice with a PDE9 selective inhibitor (PF-4181366) increased cGMP levels in the
same brain regions as well as in the cerebrospinal fluid [128]. The analysis of PDEs expres-
sion in rodent testis revealed that PDE9A mRNA is detectable in mouse testis [31] and in
Leydig cells obtained from rats [49] while expression data on human testis are controver-
sial [20,64] (Tables 1 and 2).
2.10. PDE10
PDE10 is a dual cAMP/cGMP hydrolyzing enzyme encoded by a single gene,
PDE10A present in two major variants, PDE10A1 and PDE10A2 [54,55] (Figure 10). It
shows a higher affinity for cAMP and may function in vivo as a cAMP-inhibited cGMP
PDE [129].
Figure 10. PDE10 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [130]).
PDE10A is mainly expressed in the thyroid, pituitary glands and brain; it has been
suggested as a regulator of learning and memory processes [131].
The expression of PDE10A has been investigated by Baxendale and Fraser, analyzing
the presence and function of murine PDEs and demonstrating that PDE10A transcripts
are expressed in testis but not in mature spermatozoa [31] (Table 1). Other works con-
firmed this expression paern in rodent testis with different technical approaches [54].
Among them, Andric et al. clearly demonstrated that rat Leydig cells express PDE10 tran-
script and chronic treatment with a selective Pde5i is not able to modify PDE10 mRNA
expression levels [49]. A decade before, Fujishige et al. reported a strong PDE10A im-
munoblot signal corresponding to high enzymatic activity in rat testis and striatum [132],
removing any reasonable doubt on the presence of PDE10 in these two organs.
Figure 10.
PDE10 gene family. Human variants are depicted. This figure was gathered through
analysis and cross-referencing of online databases (https://www.ensembl.org/ and https://www.
uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively.
The boxes with different colors indicate alternative exons. The maximum number of exons illustrated
is 17 and ellipsis indicate exons not shown (adapted from [130]).
PDE10A is mainly expressed in the thyroid, pituitary glands and brain; it has been
suggested as a regulator of learning and memory processes [131].
The expression of PDE10A has been investigated by Baxendale and Fraser, analyzing
the presence and function of murine PDEs and demonstrating that PDE10A transcripts are
expressed in testis but not in mature spermatozoa [
31
] (Table 1). Other works confirmed
this expression pattern in rodent testis with different technical approaches [
54
]. Among
them, Andric et al. clearly demonstrated that rat Leydig cells express PDE10 transcript and
chronic treatment with a selective Pde5i is not able to modify PDE10 mRNA expression
levels [
49
]. A decade before, Fujishige et al. reported a strong PDE10A immunoblot signal
corresponding to high enzymatic activity in rat testis and striatum [
132
], removing any
reasonable doubt on the presence of PDE10 in these two organs.
PDE10A immunoreactivity is absent in the epididymal spermatozoa of mice; however,
human spermatozoa have been demonstrated to express PDE10 [
56
]. Data on humans also
come from a microarray analysis of PDEs expression showing that PDE10A is expressed at
moderate levels in normal testis [64] (Table 2).
The exact function of PDE10 in spermatogenesis remains unclear; what is known from
preclinical studies is that its constitutive deletion does not affect sperm’s ability to fertilize
oocytes [133].
2.11. PDE11
PDE11 is the most recently identified PDE and exhibits a dual substrate specificity for
both cAMP and cGMP [
134
]. It is encoded by only one gene, PDE11A, that produces four
variants (PDE11A1–PDE11A4) displaying different amino termini [135] (Figure 11).

Int. J. Mol. Sci. 2023,24, 7617 14 of 26
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 14 of 27
PDE10A immunoreactivity is absent in the epididymal spermatozoa of mice; how-
ever, human spermatozoa have been demonstrated to express PDE10 [56]. Data on hu-
mans also come from a microarray analysis of PDEs expression showing that PDE10A is
expressed at moderate levels in normal testis [64] (Table 2).
The exact function of PDE10 in spermatogenesis remains unclear; what is known
from preclinical studies is that its constitutive deletion does not affect sperms ability to
fertilize oocytes [133].
2.11. PDE11
PDE11 is the most recently identified PDE and exhibits a dual substrate specificity
for both cAMP and cGMP [134]. It is encoded by only one gene, PDE11A, that produces
four variants (PDE11A1–PDE11A4) displaying different amino termini [135] (Figure 11).
Figure 11. PDE11 gene family. Human variants are depicted. This figure was gathered through anal-
ysis and cross-referencing of online databases (hps://www.ensembl.org/ and hps://www.uni-
prot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively. The
boxes with different colors indicate alternative exons. The maximum number of exons illustrated is
17 and ellipsis indicate exons not shown (adapted from [136]).
In humans, PDE11A is relatively highly expressed in skeletal muscle and prostate
while moderate expression levels have been detected in the testis, pituitary and thyroid
glands [137]. Data on rodents suggest a role of PDE11 in sperm development and function
since PDE11 is expressed at high levels in the testes and developing spermatozoa, and
ejaculated sperm from PDE11 knock-out mice showed lower spermatozoa counts and
lower sperm motility reflecting a compromised fertilizing capacity [31,57] (see Table 1).
Human spermatogonia, spermatocytes and spermatids as well as Leydig cells all express
PDE11 [69] (Table 2). Data on human also come from a microarray analysis of PDEs ex-
pression showing that PDE11A is present at moderate levels in normal testis [64]. Even if
presence of PDE11 in the testis is unquestionable, its effect on human sperm function is
still unclear.
3. An Overview of Phosphodiesterase Families in Testicular Tumors
3.1. Testicular Tumors
Testicular tumors are the most common solid neoplasm of young adult men between
20–40 years of age. The two principal categories of testicular cancer comprise Testicular
Germ Cells Tumors (TGCTs), which represent the majority of testis malignancies and
“non-germ cell tumors”. Gonads are the elective site where these tumors arise; however,
when the location is in extragonadal sites, they are called Extragonadal Germ Cell Tumor
[138,139]. TGCTs can be distinguished according to their histological composition (Hema-
toxylin and Eosin and immunohistochemistry staining using specific markers), the germ
cell lineage (aberrant development of the physiological germ cell at different phases of
maturation) and the age of onset (pediatric, adolescent or adult).
TGCTs can arise from the precursor lesion called germ cell neoplasia in situ (GCNIS)
that originates in fetal life. GCNIS remains dormant until puberty then, under hormonal
influences, it starts to proliferate and initiate te invasive growth. Histologically, GCNIS
Figure 11.
PDE11 gene family. Human variants are depicted. This figure was gathered through
analysis and cross-referencing of online databases (https://www.ensembl.org/ and https://www.
uniprot.org, accessed on 30 March 2023). Boxes and lines represent exons and introns, respectively.
The boxes with different colors indicate alternative exons. The maximum number of exons illustrated
is 17 and ellipsis indicate exons not shown (adapted from [136]).
In humans, PDE11A is relatively highly expressed in skeletal muscle and prostate
while moderate expression levels have been detected in the testis, pituitary and thyroid
glands [
137
]. Data on rodents suggest a role of PDE11 in sperm development and function
since PDE11 is expressed at high levels in the testes and developing spermatozoa, and
ejaculated sperm from PDE11 knock-out mice showed lower spermatozoa counts and lower
sperm motility reflecting a compromised fertilizing capacity [
31
,
57
] (see Table 1). Human
spermatogonia, spermatocytes and spermatids as well as Leydig cells all express PDE11 [
69
]
(Table 2). Data on human also come from a microarray analysis of PDEs expression showing
that PDE11A is present at moderate levels in normal testis [
64
]. Even if presence of PDE11
in the testis is unquestionable, its effect on human sperm function is still unclear.
3. An Overview of Phosphodiesterase Families in Testicular Tumors
3.1. Testicular Tumors
Testicular tumors are the most common solid neoplasm of young adult men between
20–40 years of age. The two principal categories of testicular cancer comprise Testicular
Germ Cells Tumors (TGCTs), which represent the majority of testis malignancies and “non-
germ cell tumors”. Gonads are the elective site where these tumors arise; however, when the
location is in extragonadal sites, they are called Extragonadal Germ Cell Tumor [
138
,
139
].
TGCTs can be distinguished according to their histological composition (Hematoxylin and
Eosin and immunohistochemistry staining using specific markers), the germ cell lineage
(aberrant development of the physiological germ cell at different phases of maturation) and
the age of onset (pediatric, adolescent or adult).
TGCTs can arise from the precursor lesion called germ cell neoplasia in situ (GCNIS)
that originates in fetal life. GCNIS remains dormant until puberty then, under hormonal
influences, it starts to proliferate and initiate te invasive growth. Histologically, GCNIS
transforms into seminoma or pure or mixed non-seminoma that includes embryonal cell
carcinoma, choriocarcinoma, yolk sac tumors and teratomas. Seminomas, also referred to
as Type II TGCTs, occur in adolescents and young adults (15 and 40 years of age), are the
most common form of TGCTs and are always malignant. Type I and type III TGCTs do not
arise from GCNIS and they occur in pediatric or in elderly men, respectively. Type I TGCTs
are histologically subdivided into teratomatous tumors (benign) and yolk sac tumors
(malignant). Type III TGCTs (spermatocytic seminoma, previously known as spermatocytic
seminoma) contain cells that are similar to secondary spermatocytes [140].
Non-germ cells testicular tumors include a fair variety of neoplastic diseases. Among
them, Leydig cell tumors (LCTs) represent the most common non-germ cell testicular
tumors accounting for 3–22% of all testicular neoplasms [
141
–
144
]. Given the growing use
of testis ultrasonography, but also increased exposure to endocrine disruptors [145,146], a
progressive rise in the diagnosis of LCTs has been observed [
145
,
146
]. It is widely accepted
that LCTs are always benign in the pediatric population whereas the malignant potential
increases with age, peaking around 60 years of age. Sertoli tumors, the other type of

Int. J. Mol. Sci. 2023,24, 7617 15 of 26
non-germinal testicular tumors, are extremely rare accounting for only 1% of all testicular
tumors.
3.2. Phosphodiesterases in Testicular Cancer
Neoplastic transformation can be driven by genetic and epigenetic changes, that in
turn alter signaling pathways involved in the proper control of cell division, death and
motility. cAMP and cGMP signaling participate in cell proliferation, energy homeostasis
and metabolism [
147
–
151
] and when these signals become aberrant, we can assist in the
onset of several pathological processes, including tumorigenesis [
152
,
153
]. The alteration
of cAMP/cGMP by ACs/guanylyl cyclases, respectively, have been associated with both
cyclic nucleotide synthesis or degradation by PDEs [154–158].
To identify gene signatures that may drive the development of seminoma, a gene
expression profile was performed in seminoma samples and compared to normal testis.
Chen and collaborators identified 1563 upregulated genes and 1939 downregulated genes.
Among the downregulated pathways they found several metabolic signals, such as FoxO
and Wnt, but more interestingly the cGMP-PKG signaling pathway [
159
]. Another gene
that was found to be significantly associated with testicular cancer was PDE1A [
160
] and
in vivo
exposure of mice to secondhand smoke produced a unique ‘frameshift’ variant
within the murine PDE1A suggesting an involvement of this PDE in non-familial testicular
cancer [161].
PDE11A has been identified as another genetic modifying factor for the development
of testicular tumors and it has been reported that PDE11A-inactivating variants may in-
crease the risk of developing familial and bilateral testicular germ cell tumor. The first
demonstration of this relationship comes from the observations of Horvart et al. who,
sequencing PDE11A in 95 patients with TGCTs, identified several functional variants previ-
ously implicated in adrenal tumor predisposition [
162
]. This topic was further addressed
by Azevedo et al., which reported inactivating germline mutations of PDE11A as modi-
fiers of familial testicular germ cell tumors risk. After identifying PDE11 mutations, they
transfected NTERA-2 and Tcam-2 cells with several mutated variants of PDE11A (R52T,
F258Y, Y727C, R804H, V820M, R867G and M878V). They were able to demonstrate that
cAMP levels were significantly higher, and the relative phosphodiesterase activity was
lower in PDE11 mutated cells compared to wild-type cells [
163
]. A decisive contribution
to this issue was given by the studies led by Pathak et al., indeed in a prior candidate
gene study of 94 familial testicular germ cell tumors subjects, they were able to identify a
significant correlation between the presence of functionally abnormal variants in PDE11A
and a high risk to develop familial TGCT. They proposed a subsequent broader validation
study sequencing the PDE11A coding region in 259 additional TGCT patients (both famil-
ial and sporadic) and 363 healthy controls. This analysis revealed the presence of more
than 50 PDE11A variants, two of which were functionally characterized and shown to be
functionally inactivating, resulting in reduced PDE activity and increased cAMP levels
(Figure 12) [
164
]. Recently, Faja et al. analyzed PDE11 mutational status in semen from
patients with unilateral and bilateral sporadic TGCTs and healthy controls. They were
able to detect ten polymorphisms, not previously associated with testicular cancer, that are
positively associated with TGCTs and correlating with sperm count [165].
PDEs misregulation has been reported also in non-germ testicular tumours. A higher
frequency of PDE11A sequence variants in patients with large-cell calcifying Sertoli cell
tumors was identified, pointing out how PDE11A could be considered a genetic modifying
factor for the development of testicular tumors, acting directly on germ cells or indirectly
through somatic cells [
166
]. Recently, we have demonstrated that during Leydig cell
neoplastic transformation PDE8B expression levels increased compared to the normal testis,
while PDE8A levels were almost comparable between the two sample groups suggesting,
for the first time, a potentially pivotal role of PDE8B in LCs dysfunction [65] (Figure 12).

Int. J. Mol. Sci. 2023,24, 7617 16 of 26
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 16 of 27
functionally inactivating, resulting in reduced PDE activity and increased cAMP levels
(Figure 12) [164]. Recently, Faja et al. analyzed PDE11 mutational status in semen from
patients with unilateral and bilateral sporadic TGCTs and healthy controls. They were
able to detect ten polymorphisms, not previously associated with testicular cancer, that
are positively associated with TGCTs and correlating with sperm count [165].
Figure 12. cAMP/PKA/PDEs mediated physiopathological processes in seminiferous tubules. LH:
luteinizing hormone; FSH: follicle-stimulating hormone; LHR: luteinizing hormone receptor; FSHR:
follicle-stimulating hormone receptor; Gs: G alpha subunit s protein; AC: adenylate cyclase; ATP:
adenosine triphosphate; AMP: adenosine monophosphate; cAMP: cyclic adenosine monophos-
phate; PDEs: phosphodiesterases; PKA: protein kinase A; ERK1/2: extracellular signal-regulated ki-
nase; CREB: cAMP-responsive element binding protein; CREM: cAMP-responsive element modu-
lator; ABP: androgen binding protein.
PDEs misregulation has been reported also in non-germ testicular tumours. A higher
frequency of PDE11A sequence variants in patients with large-cell calcifying Sertoli cell
tumors was identified, pointing out how PDE11A could be considered a genetic modify-
ing factor for the development of testicular tumors, acting directly on germ cells or indi-
rectly through somatic cells [166]. Recently, we have demonstrated that during Leydig cell
neoplastic transformation PDE8B expression levels increased compared to the normal tes-
tis, while PDE8A levels were almost comparable between the two sample groups suggest-
ing, for the first time, a potentially pivotal role of PDE8B in LCs dysfunction [65] (Figure
12).
4. Discussion
In view of the enormous information available on PDEs in all tissues, it surprises that
there is a remarkable paucity of studies regarding the presence, specific function and sub-
cellular location of PDE subtypes in human testis and even less information on human
testicular cancer is available.
Testis is a complex endocrine organ regulated by intra- and extra-testicular pathways
that synergistically interact. In particular, mammalian spermatogenesis involves the in-
terplay of different cell types and comprises a series of cellular and biochemical metamor-
phoses and the impairment of each step of this complex network could lead to neoplastic
transformation. The single-cell types involved have been shown to express diverse PDEs
FSH R LH R
AC
Gαs
cAM P
AMP
CREB
ERK1/2
P P
LHFSH
Gαs
PKA
PDEs
steroidogenic
signals
CREM
P
Leydig cells:
Testosterone
Serto li ce lls:
ABP
Arom atase
Growth factors
ATP
FSH R LH R
AC
Gαs
cAM P
AMP
CREB
ERK1/2
P P
LHFSH
Gαs
PKA
PDEs
impaired
steroidogenic
signals
CREM
P
ATP
Figure 12.
cAMP/PKA/PDEs mediated physiopathological processes in seminiferous tubules. LH:
luteinizing hormone; FSH: follicle-stimulating hormone; LHR: luteinizing hormone receptor; FSHR:
follicle-stimulating hormone receptor; Gs: G alpha subunit s protein; AC: adenylate cyclase; ATP:
adenosine triphosphate; AMP: adenosine monophosphate; cAMP: cyclic adenosine monophosphate;
PDEs: phosphodiesterases; PKA: protein kinase A; ERK1/2: extracellular signal-regulated kinase;
CREB: cAMP-responsive element binding protein; CREM: cAMP-responsive element modulator;
ABP: androgen binding protein.
4. Discussion
In view of the enormous information available on PDEs in all tissues, it surprises
that there is a remarkable paucity of studies regarding the presence, specific function and
subcellular location of PDE subtypes in human testis and even less information on human
testicular cancer is available.
Testis is a complex endocrine organ regulated by intra- and extra-testicular path-
ways that synergistically interact. In particular, mammalian spermatogenesis involves the
interplay of different cell types and comprises a series of cellular and biochemical metamor-
phoses and the impairment of each step of this complex network could lead to neoplastic
transformation. The single-cell types involved have been shown to express diverse PDEs
and their localization and compartmentalization contribute to a spatiotemporal regulation
of cAMP or cGMP [167].
cAMP-dependent signaling pathway, and to a lesser extent, cGMP signaling, are the
main molecular mechanisms that played a major role in orchestrating the expression of the
many genes in spermatogenesis [168–171].
Leydig cells have a crucial role in the regulation of steroidogenesis and spermato-
genesis, since they are the production site of testosterone, which has a main role in fetal
development and maturation, while the growth and differentiation of germ cells (i.e., the
precursors of sperm) require Sertoli cells [
172
,
173
]. The synthesis and release of steroid
hormones from Leydig cells, and the maturation of Sertoli cells, happen in response to
two pituitary gonadotropins, LH and FSH. LH and FSH bind to the LH receptors and
FSH receptors on Leydig and Sertoli cells, respectively, stimulating AC activity that raises
intracellular cAMP level and activates PKA which regulates the expression of genes related
to the steroidogenesis [
174
]. Substantial evidence for the regulatory function of PDEs in
Leydig cells has been reported as a stimulatory effect of a pan-PDEi on testosterone release

Int. J. Mol. Sci. 2023,24, 7617 17 of 26
by primary LCs [
175
], indicating that one or more PDEs might be active in Leydig cells to
modulate the intensity, duration and the desensitization of the LH-stimulated hormonal
response [
175
] (Figure 12). Indeed, it was demonstrated that Leydig cells express transcripts
for several cAMP-specific PDEs (Tables 1and 2) most of them contributing to Leydig cell
response through LH receptor-cAMP signaling [
52
,
176
]. Testosterone is able to increase
PDE5A, PDE6D and PDE9A expression [
176
]. Increased levels of cGMP in Leydig cells
isolated from testosterone-treated rats confirmed an active role of cGMP-specific PDEs in
degrading nitric oxide-stimulated cGMP [49].
Within the tubules, cGMP and cAMP cooperate in controlling germ cell differentiation,
both directly and indirectly through Sertoli cells. cAMP-dependent signal transduction
pathway, in particular, is one of the major regulatory mechanisms that operates at different
stages of spermatogenesis. The regulation of gene expression is exerted via a family
of nuclear transcription factors that bind to a specific DNA element designated cAMP-
response element (CRE). The two predominant members of this family are the cAMP-
response element binding protein (CREB) and the cAMP-responsive element modulator
(CREM), which, in turn, transactivate the transcriptional expression of cAMP-responsive
target genes [
177
,
178
]. The importance of CREM to male fertility was evident through
the study of CREM knockout male mice, which are sterile having absolutely no mature
spermatozoa [
179
], whereas CREB-mediated survival factor/s produced by Sertoli cells
protect germ cell survival [
180
], strongly arguing for the essential role of CREB/CREM in
sperm development in humans. Sperm function during capacitation, such as activation of
motility, changes in the motility pattern known as hyperactivation have been attributed to
the modulation of cAMP/cGMP [181].
Since 2017, mammalian testis transcriptomes, at different developmental stages and in
both physiological and pathological conditions, have been extensively studied at the single-
cell level by using single-cell RNA-sequencing (RNA-seq) revealing cell transcriptomes
heterogeneities at a high resolution [
182
–
194
]. PDE transcripts do not appear as the top
expressed gene or the most differentially expressed gene. Even though RNA-seq is a
powerful tool it possesses some pitfall, it induces, for example, great RNA loss and low
sequencing depth making it difficult to capture low abundant RNAs [
195
], thus causing
an information loss. More information that is lost during RNAseq regards RNA isoform
variants. Given that the testis is one of the organs that mostly exploit the potential of
alternative splicing (AS) [
196
] and that, as mentioned, PDEs undergo extensive AS, it is
conceivable that their transcripts have been underestimated. Moreover, it should be taken
into account that, in germ cells, a temporal gap between mRNA transcription and protein
synthesis exists, in part due to the fact that RNA synthesis terminates long before the
spermatids complete their differentiation; therefore, it is mandatory to verify presence,
specific function and subcellular location of the translated PDEs. Finally, a similar but not
totally overlapping PDE expression pattern in testis between human and mouse tissues
has been revealed. Taking this in mind, we demonstrated that in human testis, PDE8A
and PDE8B localized in the cytosol in granular structure in Leydig cells as in mouse testis
but more interestingly that PDE8A is expressed in round spermatids close to acrosome,
and that it associates with the trans-Golgian region in specific stages, suggesting that it
supports and sustains the trafficking of the vesicles originating from the Golgi apparatus
for the acrosome biogenesis. We are encouraging a more systematic analysis of PDEs
role/localization in the human testis to exploit unexpected functions of these enzymes.
Interference of cAMP/cGMP signaling pathway has been shown to be linked to tumori-
genesis [
155
,
197
] and PDEs overexpression has been already described in several cancer types
such happened for PDE11A and PDE8B, in adrenal hyperplasia/adenomas
[162,198,199]
.
Indeed some PDEs have been already proposed as a potential biomarker for different
tumoral contests [
200
–
204
] and we have proposed PDE8B as a promising biomarker for
Leydig cell tumours. We believe that multi-omics will be useful to deeply analyze PDEs
expression and activity for the translation of such findings in clinical practice. Moreover,
PDE-opathies name has been coined to identify a set of disorders caused by germline

Int. J. Mol. Sci. 2023,24, 7617 18 of 26
mutations of PDEs [
205
], it would be interesting to extend this concept to testicular cancers
with a more focused screening since their pharmacological inhibition has been proposed
as an anticancer strategy in several tumors [
18
], but we are still far from the “they lived
happily ever after”.
Author Contributions:
Conceptualization, F.B.; writing—original draft preparation, F.B., F.C. and
M.R.A.; visualization, M.R.A.; writing—review and editing, F.B., F.C. and M.A.V.; All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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