**3. PCSK4 as a potential sperm antigen for ASA contraceptive developments**

## **3.1 Biochemistry of PCSK4**

One sperm antigen potential for development of contraceptive ASA is the proprotein convertase subtilisin/kexin type 4 (PCSK4) [78–81]. PCSK4 is an enzyme protein expressed in abundant on the outer surface of acrosomal plasma membrane of mammalian spermatozoa (**Figure 1**). It has a molecular weight of 45 kDa [79, 83, 84]. Biochemically it plays role in the proteolytic activation of precursor proteins in the cellular secretory pathway and physiologically it has a specific important role in mammalian reproductive process [79, 83–85]. PCSK4 gene is a 9 kilobase (kb) DNA, consists of 15 exon and 14 intron which in humans it is located in the chromosome 19 [78, 80, 85]. It is a protein associated with bacterial subtilisin and yeast kexin where its biosynthesis takes place in endoplasmic reticulum in the form of a multi-domain preprotein [78, 80]. PCSK4 is one among nine family members of calcium-dependent serin endoproteinase *Proprotein Convertase Subtilisin/Kexin* (PCSK) [79, 83–85]. These nine family members of PCSK are: PCSK1 and PCSK2 which are expressed in endocrine and neuroendocrine cells; PCSK3, PCSK5, PCSK6 and PCSK7 are widely expressed by different types of cells; PCSK4 is specifically presented by gonadal and placental cells and; PCSK8 and PCSK9 which play roles in the synthesis pathways of cholesterol and lipid acid [79, 80, 86–88]. PCSK 1 to 7 belong to sub-family of kexin while PCSK8 and PCSK9 belong to sub-family of pirolysin and proteinase-K, respectively [79, 80, 86]. PCSK 1 to 3 are also known

*Innovations in Assisted Reproduction Technology*

intradermally in female mice caused anti fertility effects [53].

and all of these immune-converted monkeys were sterile [63].

eggs develop into blastocysts when intact mice is used [64].

Heparin-binding serpin, protein C inhibitor (PCI), is a nonspecific serpin that inactivates many plasmatic and extravascular serine proteases. Mutant male mice lacking PCI gene are infertile but apparently healthy. Histologic examination showed that Sertoli cells and their barrier were destroyed. The resulting sperm are malformed, lack of tail and deformed head and immobilised, similar to those seen in some cases of men infertility [64], thus the effect of PCI on testicle tissues is apparently cytotoxic. In *in-vivo* fertilisation experiments, only 0.5% (n = 416) eggs are fertilised by sperm of mutant male compared to the rate of 92% (n = 415) of

The 80 kDa human sperm antigen (HAS) is known to present on the head of the human and rat spermatozoa, in the testes and epididymis but not in other somatic tissues. Active immunisation of male and female rats with 80 kDa HSA caused infertility in all the immunised animals [65]. Further, active immunisation of male rabbits, rat and marmosets with synthetic peptide-1 of HSA induced reversible infertility in 100, 100% and 6 of 7 of the respective animals [66]. It strongly indicates that antibodies anti-HSA have potential as immuno-contraceptive agents. Additionally, anti-HSA antibody induced *in-vitro* agglutination of human, rat and

**2.4 Various ASA that immobilise sperm**

tration-dependently inhibits human sperm acrosome reaction [58].

Two sperm antigens have been studied for this property and for their potential to base a development of contraceptive agents; YLP12 and testis specific antigen-1 (TSA-1). A dodecamer sequence designated YLP12 is a peptide sequence that have been identified to specifically localise on the acrosome and tail of spermatozoa [55]. It is known to recognise ZP3 component of human ZP proteins, to involve in sperm-ZP binding and the antibodies against synthetic 12-mer peptide based on YLP12 sequence was reported to specifically inhibit human sperm-ZP binding [56]. The antibody anti-YLP12, in other study, was reported to show a concentrationdependent inhibition of acrosome reaction but did not affect the sperm motility [57]. Immunisation of murine model with synthetic YLP12 produced antibodies affected fertility by reducing sperm capacitation, acrosome reaction and spermoocyte binding in an *in-vitro* assay but, immunised murine remained fertile and were capable of delivering the equal number of pups compared to control [55]. In contrast, other study reported that a sperm-specific YLP12 DNA vaccine injected

A study reported that TSA-1 is localised to the regions of acrosome, equatorial, mid-piece and tail of human sperm. An *in-vitro* test discovered that TSA-1 concen-

Sperm tail is widely known as an organelle of motility. Proteomic study showed that proteins extracted from tail fraction of sperm can be classified according to their functions into two main groups: proteins related to metabolism and energy production from endogenous sources, and those related to tail structure and motility [59]. These groups however, are at the end function together to support sperm motility. *Ep*ididymal *p*rotease *in*hibitor (Eppin) is expressed in the testis and epididymis tissue and on the acrosome and tail of human sperm [60–62]. An in-vitro assay showed that the monkey anti-eppin antibodies decreased the progressive motility of human spermatozoa in terms of distance travelled and speed [62]. Other study suggested that blockade of Eppin epitope by anti-eppin antibody would halt the acrosome reaction through reduction of ionophore-induced calcium influx [60]. Further pre-clinical trial was performed *in-vivo* in non-human primates and showed that Eppin immunised *Macaca radiate* developed high titers to Eppin (78%)

**224**

monkey sperm [67].

### **Figure 1.**

*Using monoclonal antibody anti-human PCSK4 and rodamin as staining, PCSK4 molecules are detected in abundant on the membrane surface of the head of human spermatozoa under* Laser Scanning Microscope *(LSM) examination [82].*

as, consecutively, PC1/3, PC2 and furin, while PCSK 5 to 9 are also recognised as PC5/6, PACE4, PC7, SKI-1/SIP and NARC-1, respectively [89].

PCSK4 is synthesised as a zymogen, proPCSK4, in the endoplasmic reticulum [81]. Its maturation process remains unclear but suggested to be by an autocatalytic process that cleaves the molecule at two sites situated between prodomain and the catalytic domain [80]. PCSK4 proprotein contains five domains located consecutively, i.e. the signal peptide (SP) domain, prodomain, catalytic domain, a domain of 150 amino acids called P (Protease) domain or homo B domain and the C-terminal domain which carries transmembrane domain [80]. The SP domain of PCSK4 contains signal peptide which directs precursor proteins toward the secretory pathway. The Prodomain acts as an intra-molecule chaperone and regulator of the catalytic activity of the enzyme, removal of this domain is essential for the activity of the enzyme [90]. The primary and secondary cleavage sites of proPCSK4 are situated between prodomain and catalytic domain. Catalytic domain of PCSK4 is the active site of the enzyme and carries Asp-His-Ser catalytic triad characteristics of serin proteinase [80].

The P domain of PCSK4 plays an important role for proper folding, contains beta fold sandwich of galactose bond domain capable of mediating carbohydrates, phospholipids or membrane receptors. This P domain acts as a regulator site of ultimate enzyme activities under influences of optimum pH and calcium, but also in stabilising the structure of catalytic domain [80, 90]. It helps to balance asymmetric surface at the region of catalytic domain bond, owing to the specific characteristic of multi-basic residues of this enzyme (the consensus of substrate order is Arg-X-Lys-Arg-Arg, where X can be an amino acid; the site of peptide dihydrolysis) [80, 83, 91]. The C-terminal variable domain influences intracellular localisation, in-out recycle of proteins and protein–protein interactions. N-glycosylation sites are located at P domain and at C-terminal domain [80].

Expressions of PCSK4 are discovered in abundant in reproductive organs, especially in testis. In testis, they can be found in epididymic and germinal cells i.e. in acrosomal granules of round spermatids, in acrosomal ridges of elongated spermatids and on the acrosomal plasma membrane of spermatozoa [80, 81, 84, 92]. The PCSK4 or named as proprotein convertase PC4 in a study, is also expressed in the human placenta and macrophage-like cells in the ovary [87, 88].

### **3.2 Physiological roles of PCSK4**

Intracellularly, PCSKs takes action in the limited endoproteolytic regulation mechanism of the secretory pathway. Limited endoproteolysis constitutes the

**227**

*Human Contraceptives: Current Status, Sperm Antigen Inhibitors and an Insight into PCSK4*

post-translational modifications of proteins by which cells diversify and regulate gene products [78]. Endoproteolytic process occurs during modifications to activate many precursor proteins in biological cell functions including zymogen activations, formations of peptide hormones, complement activations, blood clotting and blood clot lysing, angiogenesis and tissue re-modelling. Secretory pathway processes of eukaryotic cells are mainly assisted by carboxyl residues of Lys or Arg (P1) in the order R/K – (X)n – X/K/R – R (where: X = amino acids other than Cys; n = 1, 3, or 5;

PCSK4 present in acrosome region of sperm thus, is hypothesised to play role in capacitation and acrosome reaction [80, 84]. SPCSK4-null sperm has normal appearance, normal motility competence and undergo normal capacitation process but, following capacitation they suffer reduced hyperactivated motility [93]. Postcapacitation hyperactivity is a qualitative characteristics of sperm which thought to be important to assist sperm penetrating zona pellucida of an egg. PCSK4-null sperms show a reduced fertilisation competence *in-vitro* and the embryo resulted

Two molecules have been known as the natural substrates of PCSK4; propituitary adenylate cyclase-activating protein (proPACAP) and Insulin-like growth factor II (IGF-II) [87, 94]. The proPACAP has two active isoforms; PACAP38 and PACAP27 residues. PACAPs are expressed in hypothalamus, in extra-hypothalamic regions of the brain, in the granulosa cells of the developing ovarian follicles of the rat and transiently in rat spermatid cap but, are absent at the other stages of spermatogenesis. They are also expressed in Sertoli cells and Leydig cells. Gonadal PCSK4 is the only enzyme that activates proPACAP both in the testis and the ovary of the mice [94]. A study reported that PACAP null female mice failed to implant its embryo to the uterus [95], suggesting proPACAP activation conducted by PCSK4 is pivotal in embryonic implantation. PCSK4 null male mice lack of PACAP activation and produce normal but incompetent sperm. It leads to the hypothesised that PACAP may not important in maturation process of the sperm but involves in the

production of molecules required for the functional mature sperm [94].

Other substrate of PCSK4, IGF-II, is discovered in placenta and its inactivity has been shown to be involved in the pathophysiology of intrauterine growth restriction (IUGR) of human foetus; a major cause of perinatal death. In this pathway, Placental PCSK4 activates pro-IGF-II to form a half-matured IGF-II and successively mature IGF-II as a result of the cleavage of its terminal basic residues by carboxypeptidases [87]. Inhibition of Placental PCSK4 by a PCSK4-specific inhibitor blocks pro-IGF-II processing resulting a reduced trophoblast cell migration [87], likely due to reduced effectivity of trans-placental diffusional exchange leading to reduced nutritional supply [96]. The locality and functionality of the two substrates of PCSK4 indicate that PCSK4 are physiologically important during zona penetration and embryonic development.

Studies in model animals showed that individuals with PCSK4 expression disorder have significantly lower fertilisation capability [79]. In our study, intact male *Rattus norvegicus* previously injected with antibody anti-PCSK4 was allowed to fertile female rats and it showed that the number of off-springs delivered by these female rats significantly declined in accordance with the increased doses of antibody anti-PCSK4 injected [82]. These indicate that inactivation of PCSK4 by

*DOI: http://dx.doi.org/10.5772/intechopen.89721*

K or R = the place of P2 amino acid) [78, 86, 91].

from PCSK4-null sperm fails to develop further [93].

**3.3 Substrates of PCSK4**

**3.4 Inhibitors of PCSK4**

injectable anti-PCSK4 could prevent conception.

*Human Contraceptives: Current Status, Sperm Antigen Inhibitors and an Insight into PCSK4 DOI: http://dx.doi.org/10.5772/intechopen.89721*

post-translational modifications of proteins by which cells diversify and regulate gene products [78]. Endoproteolytic process occurs during modifications to activate many precursor proteins in biological cell functions including zymogen activations, formations of peptide hormones, complement activations, blood clotting and blood clot lysing, angiogenesis and tissue re-modelling. Secretory pathway processes of eukaryotic cells are mainly assisted by carboxyl residues of Lys or Arg (P1) in the order R/K – (X)n – X/K/R – R (where: X = amino acids other than Cys; n = 1, 3, or 5; K or R = the place of P2 amino acid) [78, 86, 91].

PCSK4 present in acrosome region of sperm thus, is hypothesised to play role in capacitation and acrosome reaction [80, 84]. SPCSK4-null sperm has normal appearance, normal motility competence and undergo normal capacitation process but, following capacitation they suffer reduced hyperactivated motility [93]. Postcapacitation hyperactivity is a qualitative characteristics of sperm which thought to be important to assist sperm penetrating zona pellucida of an egg. PCSK4-null sperms show a reduced fertilisation competence *in-vitro* and the embryo resulted from PCSK4-null sperm fails to develop further [93].

### **3.3 Substrates of PCSK4**

*Innovations in Assisted Reproduction Technology*

**Figure 1.**

*(LSM) examination [82].*

as, consecutively, PC1/3, PC2 and furin, while PCSK 5 to 9 are also recognised as

*Using monoclonal antibody anti-human PCSK4 and rodamin as staining, PCSK4 molecules are detected in abundant on the membrane surface of the head of human spermatozoa under* Laser Scanning Microscope

and carries Asp-His-Ser catalytic triad characteristics of serin proteinase [80]. The P domain of PCSK4 plays an important role for proper folding, contains beta fold sandwich of galactose bond domain capable of mediating carbohydrates, phospholipids or membrane receptors. This P domain acts as a regulator site of ultimate enzyme activities under influences of optimum pH and calcium, but also in stabilising the structure of catalytic domain [80, 90]. It helps to balance asymmetric surface at the region of catalytic domain bond, owing to the specific characteristic of multi-basic residues of this enzyme (the consensus of substrate order is Arg-X-Lys-Arg-Arg, where X can be an amino acid; the site of peptide dihydrolysis) [80, 83, 91]. The C-terminal variable domain influences intracellular localisation, in-out recycle of proteins and protein–protein interactions. N-glycosylation sites are

Expressions of PCSK4 are discovered in abundant in reproductive organs, especially in testis. In testis, they can be found in epididymic and germinal cells i.e. in acrosomal granules of round spermatids, in acrosomal ridges of elongated spermatids and on the acrosomal plasma membrane of spermatozoa [80, 81, 84, 92]. The PCSK4 or named as proprotein convertase PC4 in a study, is also expressed

Intracellularly, PCSKs takes action in the limited endoproteolytic regulation mechanism of the secretory pathway. Limited endoproteolysis constitutes the

in the human placenta and macrophage-like cells in the ovary [87, 88].

located at P domain and at C-terminal domain [80].

**3.2 Physiological roles of PCSK4**

PCSK4 is synthesised as a zymogen, proPCSK4, in the endoplasmic reticulum [81]. Its maturation process remains unclear but suggested to be by an autocatalytic process that cleaves the molecule at two sites situated between prodomain and the catalytic domain [80]. PCSK4 proprotein contains five domains located consecutively, i.e. the signal peptide (SP) domain, prodomain, catalytic domain, a domain of 150 amino acids called P (Protease) domain or homo B domain and the C-terminal domain which carries transmembrane domain [80]. The SP domain of PCSK4 contains signal peptide which directs precursor proteins toward the secretory pathway. The Prodomain acts as an intra-molecule chaperone and regulator of the catalytic activity of the enzyme, removal of this domain is essential for the activity of the enzyme [90]. The primary and secondary cleavage sites of proPCSK4 are situated between prodomain and catalytic domain. Catalytic domain of PCSK4 is the active site of the enzyme

PC5/6, PACE4, PC7, SKI-1/SIP and NARC-1, respectively [89].

**226**

Two molecules have been known as the natural substrates of PCSK4; propituitary adenylate cyclase-activating protein (proPACAP) and Insulin-like growth factor II (IGF-II) [87, 94]. The proPACAP has two active isoforms; PACAP38 and PACAP27 residues. PACAPs are expressed in hypothalamus, in extra-hypothalamic regions of the brain, in the granulosa cells of the developing ovarian follicles of the rat and transiently in rat spermatid cap but, are absent at the other stages of spermatogenesis. They are also expressed in Sertoli cells and Leydig cells. Gonadal PCSK4 is the only enzyme that activates proPACAP both in the testis and the ovary of the mice [94]. A study reported that PACAP null female mice failed to implant its embryo to the uterus [95], suggesting proPACAP activation conducted by PCSK4 is pivotal in embryonic implantation. PCSK4 null male mice lack of PACAP activation and produce normal but incompetent sperm. It leads to the hypothesised that PACAP may not important in maturation process of the sperm but involves in the production of molecules required for the functional mature sperm [94].

Other substrate of PCSK4, IGF-II, is discovered in placenta and its inactivity has been shown to be involved in the pathophysiology of intrauterine growth restriction (IUGR) of human foetus; a major cause of perinatal death. In this pathway, Placental PCSK4 activates pro-IGF-II to form a half-matured IGF-II and successively mature IGF-II as a result of the cleavage of its terminal basic residues by carboxypeptidases [87]. Inhibition of Placental PCSK4 by a PCSK4-specific inhibitor blocks pro-IGF-II processing resulting a reduced trophoblast cell migration [87], likely due to reduced effectivity of trans-placental diffusional exchange leading to reduced nutritional supply [96]. The locality and functionality of the two substrates of PCSK4 indicate that PCSK4 are physiologically important during zona penetration and embryonic development.

### **3.4 Inhibitors of PCSK4**

Studies in model animals showed that individuals with PCSK4 expression disorder have significantly lower fertilisation capability [79]. In our study, intact male *Rattus norvegicus* previously injected with antibody anti-PCSK4 was allowed to fertile female rats and it showed that the number of off-springs delivered by these female rats significantly declined in accordance with the increased doses of antibody anti-PCSK4 injected [82]. These indicate that inactivation of PCSK4 by injectable anti-PCSK4 could prevent conception.

The use of synthetic PCSK4 inhibitors could also serve as an option for contraception. A synthetic inhibitor of PCSK has been developed based on the knowledge, that prodomain removal is essential for activation of the enzyme. Binding of this domain to PCSK4 active enzyme could hypothetically inactivate the enzyme. A peptide, mimicking prodomain sequence near its primary activation site, was engineered and an *in-vitro* assay using a recombinant PCSK4 showed that PCSK4 mediated proteolysis was efficiently blocked by synthetic prodomain rPC4101–116 peptide [90].

Other potent synthetic PCSK4-inhibitors: tetrapeptide chloromethyl ketone and the Dec-RVKR/K-cmk (Decanoyl-RVKR/K-chloromethyl ketone) were reported to inhibit PCSK4 more potently than synthetic prodomain rPC4101–116 peptide [90]. Another substance, synthetic enediyne amino acid containing peptides, was developed and reported to inhibit PCSK4 activity *in-vitro* [97]. Further, dimeric form of CRES was reported to moderately block the PCSK4 activity to human proIGF-2 in human placental trophoblast cell line [98].

### **4. Future development of ASA contraceptives**

Only a few of aforementioned ASA underwent animal models *in-vivo* study. Among are EQT, FA-1, YLP12, *Eppin*, PCI, HSA, AKAP 4 and PCSK4. None of these molecules however, shows full inhibition of fertilisation in animal models, whereas YLP12 contraceptive studies reported various results from partial to lack of inhibition of fertility to animal models [37, 53, 63–65, 82] [53, 55, 70]. Therefore, further efforts are needed to make ASA contraceptive become reality.

Future development of ASA contraceptives might include the studies of underexplored proteins such as those involve in the energy production in the mitochondria of sperm. Proteomic approach assistance, in this regard, have enabled the discovery of large number of novel proteins [59] and allows further investigations of single protein of interest.

On the other hand, as the usage of single molecule have been impractical, alternative of future ASA development might include the study of efficacy of multivalent vaccines, in order to boost the final effective contraception effect as well as reducing potential toxic effect of high dose administration of a single substance. Study of multivalent vaccine targeting proteins of acrosomal sperm has been actually initiated. In the study, immunised monkeys recognised the five antigens used: ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, with the highest IgG average absorbance values were to ESP, SAMP 32 and SP-10 but at IgG lower values for SLLP-1 and SAMP 14 [99]. Further, capacitated sperm treated with sera from immunised monkeys showed fusion inhibition but only in two of five individuals [99]. This was a sound study that used proteins which were pre-tested to not cross react to each other. But the study used molecules that never had undergone *in-vivo* study so individual effect of the vaccine component was unknown. In addition, a study of FA-1 and YLP12 mixed vaccine in mice further support the more potential of multivalent vaccine in inducing contraceptive effects compared to monovalent vaccine [54].

The *in-vivo* study of anti-PSCK4 administration demonstrated in our laboratory indicates that it is possible to efficaciously administer contraceptive agents in the form of antiserum rather than injecting them to an individual for a few times as immunisation [82]. In the future, this approach might be more practical if the molecule could reach therapeutic concentration in the seminal plasma once the semen ejaculated thus well mixed with sperm along their journey to egg, mimicking *in-vitro* incubation of sperm with its inhibitors prior to a fertilisation challenge with eggs.

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*Human Contraceptives: Current Status, Sperm Antigen Inhibitors and an Insight into PCSK4*

Topical applications such as intravaginal administration might be strategy of choice in the antisperm contraceptive application especially for molecules which have been known to be toxic when administered systemically such as etomoxir. Topical intra-ampullar oviduct administration of antisperm agent in animal model has proven that topical ASA contraceptive application can be efficacious [37].

Existing contraceptive methods have been widely practiced by couples globally. However, some side effects limit the broader utilisation. Although ASA is a promising method of contraception it is not yet practical. More studies should be done in

Authors would like to thank to Biochemistry Laboratory of University of Brawijaya for the provision of facilities that allows the writing of this manuscript.

We authors declare that there is no conflict of interest related to the preparation

and Aulanni'am Aulanni'am3

1 Sub Division of Urology, Faculty of Medicine, Zainoel Abidin Hospital, Syiah

3 Laboratorium of Biochemistry, Faculty of Veterinary Medicine, University of

© 2019 The Author(s). Licensee IntechOpen. This chapter is 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,

2 Laboratory of Veterinary Public Health, Faculty of Veterinary Medicine,

\*

order to enable ASA to replace common contraceptive methods.

*DOI: http://dx.doi.org/10.5772/intechopen.89721*

**5. Conclusions**

**Acknowledgements**

**Conflict of interest**

of this manuscript.

**Author details**

, Widi Nugroho2

University of Brawijaya, Malang, East Java, Indonesia

Kuala University, Banda Aceh, Indonesia

Brawijaya, Malang, East Java, Indonesia

provided the original work is properly cited.

\*Address all correspondence to: aulani@ub.ac.id

Dahril Dahril1

*Human Contraceptives: Current Status, Sperm Antigen Inhibitors and an Insight into PCSK4 DOI: http://dx.doi.org/10.5772/intechopen.89721*

Topical applications such as intravaginal administration might be strategy of choice in the antisperm contraceptive application especially for molecules which have been known to be toxic when administered systemically such as etomoxir. Topical intra-ampullar oviduct administration of antisperm agent in animal model has proven that topical ASA contraceptive application can be efficacious [37].
