Germ cells are the vehicles for transmitting genetic materials to the next generation. Fully functional oocytes and sperms are therefore critical for reproduction.
Infertility is a worldwide reproductive health problems, in which some infertility cases are caused by suboptimal quality of sperm and oocytes. Oocyte maturity and quality are key limiting factors in female fertility. Several studies indicated oocyte maturity and quality will affect subsequent embryo development or pregnancy [1-3]. On the other hand, ejaculated sperm are necessary to swim and reside in the female reproductive tract for a period of time to acquire the ability to fertilize the oocyte. Many events are involved in this process, such as capacitation and acrosome reaction. Only one capacitated sperm can penetrate the zona pellucida, and then fertilize the oocyte. In this study, we purified and characterized the SERPINE2 protein from seminal plasma where capacitation moderators reside. I will describe how the SERPINE2 protein affects sperm function and oocyte maturation.
Capacitation is a complex process first independently described and defined by Chang MC et al [4] and Austin CR et al [5]. It is a physiological change in sperm that occurs in the oviduct of some mammals to acquire the ability to fertilize an egg [6]. It can be mimicked in vitro in specifically defined medium [4,6]. Our current knowledge of capacitation largely originates from in vitro studies [7-11]. Capacitation is initiated by removal of cholesterol from the sperm plasma membrane [7,8,12-14]. Cholesterol efflux leads to changes in the membrane structure and fluidity, which then increases the permeability of sperm to calcium (Ca2+) and bicarbonate (HCO3- ) ions, thus raising levels of sperm intracellular calcium ions ([Ca Ca2+ ]i) and the pH. Elevated levels of sperm intracellular Ca2+ and HCO3- can activate adenyl cyclase and lead to the increases in intracellular levels of cAMP, activation of cAMP-dependent protein kinase (PKA), and finally induction of tyrosine phosphorylation of a subset of sperm proteins [9].
Sperm capacitation usually occurs in the mammalian oviduct at the right time, but the occasional premature capacitation that may affect sperm fertility. Therefore, decapacitation factor can modulate and protect fertilizing ability of sperm, which make sure sperm do the right thing in the right place at the right time.
The existence of decapacitation factors, which are removed from the sperm head surface during the capacitation process and are able to reverse sperm capacitation, in seminal plasma have been known for more than 50 years [15]. Since the discovery of
decapacitation factors existence, many proteins have been suggested to be decapacitation factors. The sperm’s surface is immersed in a protein-rich solution of seminal plasma which is mixed with secretions from the accessory sexual glands. These seminal proteins interact with sperm and modulate changes in the sperm’s physiology, and thus prevent them from premature capacitation [16]. The decapacitation factors, which cause capacitated sperm to lose the ability to fertilize, are present in seminal plasma [15]. They are removed from the surface of the sperm’s head before or during the capacitation process. However, their identity and functions have not been fully characterized.
1.3 Identification and Characterization of Seminal Plasma Secretions Importance for
Decapacitation Factor
Most of the decapacitation factors identified so far are purified from seminal plasma secretion. The seminal vesicle is a male accessory sexual gland found in many mammalian species, which secretes a fluid called seminal vesicle secretion (SVS). SVS contributes a major portion to the liquid part of seminal plasma, which is a complex biological fluid formed from a mixture of secretions from various male reproductive tissues. Studies found that the removal of the seminal vesicle from mice and rats greatly
fertility. Proteins from seminal plasma interact with sperm and modify the sperm’s surface membrane, an essential process in maintaining sperm viability, thus modulating their functions [19]. In humans, several potential decapacitation factors that have been reported which include glycodelin-S [20], semenogelin I [21], a 130-kDa glycoprotein [22], and some mannosyl glycopeptides [23]. Several potential decapacitation factors from rodents were also identified, including a 40-kDa glycoprotein [24], phosphatidylethanolamine-binding protein 1 (PEBP1) [25]; three epididymal proteins, a cysteine-rich secretory protein 1 [26]; an acrosome-stabilizing factor [27]; and an epididymis-specific secretory protein, HongrES1 (symbol not official) [28,29]; and two secreted seminal vesicle proteins, SVA [30] and SVS2 [31,32]. Attempts have also been made to reveal sperm physiology modulating activities from other murine seminal vesicle secretory proteins. For example, a carcinoembryonic antigen-related cell adhesion molecule (CEACAM10) [33] and SVS7 [34] were found responsible for enhancing sperm motility. A kazal-type serine protease inhibitor (SPINK3), named P12, is able to suppress Ca2þ uptake by sperm [35]. Recently, the secreted LY6 protein (SSLP-1) was found to be expressed predominantly in SVS [36], although its function is unclear. In order to identify and characterize more factors important for decapacitation activities, we purified and characterized of the decapicitation factors in the mouse seminal plasma. In our previous study, we found a secreted serine protease inhibitor
Kazal-type-like (SPINKL) protein. The SPINKL protein was purified from mouse seminal vesicle secretions through a series of steps, including ion-exchange chromatography on a diethylaminoethyl-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a Q strong anion exchange column. The SPINKL protein is able to bind onto sperm and enhance sperm motility. Also, it was able to suppress BSA-stimulated sperm capacitation and block sperm-oocyte interactions in vitro, suggesting that SPINKL may be a decapacitation factor [37].
However, other important components of the SVS remain to be identified. Further investigation of the SVS proteome may enhance our understanding of normal and abnormal male reproductive physiology. We therefore performed another round of screening based on decapacitation activity in order to identify novel decapacitation factors. We purified SERPINE2 from mouse seminal vesicle secretions, based on its potential function in actively inhibiting capacitation process. I will describe our characterization of SERPINE2’s involvement in sperm decapacitation in Chapter 2.
1.4 SERPINE2 Protein Characteristics and Functions
SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor superfamily. It has broad antiprotease activity specific to serine
(PLAU) [38], and prostasin (PRSS8) [39]. SERPINE2 can inhibit PLAU and tissue-type plasminogen activator (PLAT). Plasminogen activators (PAs) are involved in tissue remodeling by converting abundant extracellular plasminogen into active protease plasmin, which degrades almost all matrix proteins [40]. The PA system is associated with many physiological processes, including ovulation, embryogenesis, and embryo implantation in female reproductive tissues [40,41], and pathological processes, such as neoplasia [40]. Two PA types, PLAT and PLAU, and four types of SERPINs, including SERPINA5, SERPINB2, SERPINE1, and SERPINE2, constitute the PA system [40].
Understanding how serpins modulate PLAT/PLAU proteolytic activities is considerably important in developing therapeutic strategies for PA-involved tissue remodeling.
1.5 The Potential Role of SERPINE2 in Reproduction
SERPINE2 protein is extensively expressed in reproductive tissues, e.g., the placenta [42,43], uterus [43,44] and ovary [45,46]. However, different species have different expression patterns of Serpine2/SERPINE2. Lin et al. reported that expression levels of SERPINE2 in the monkey endometrium and placenta during early pregnancy were weak or below the level of detection [47]. On the contrary, SERPINE2 was highly expressed in the human placenta throughout pregnancy [48]. In rats, Serpine2 mRNA
6.5 postcoitally, suggesting that it may be involved in the implantation process [49].
Our previous study demonstrated that SERPINE2 was a major PAs inhibitor in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation. It may participate in the PA-modulated tissue remodeling process in the mouse placenta and uterus [44]. PAs are associated with many reproductive processes, e.g., ovulation [50,51], embryonic development [52], embryo implantation [53], and pathological processes [40]. The expression and activity of PLAT and PLAU were detected in female reproductive tissues, including the endometrium during cycling [54,55], implantation [53], and placentation [56-59]. Also, PLAU was found to be expressed during mouse placental development [59].
1.6 Cumulus Expansion and Oocyte Maturation
In most mammals, cumulus cells are specialized granulosa cells surrounding, touching the oocyte and nourishing the oocyte development. Cumulus cells surround the oocyte to form a cumulus-oocyte complex (COC) and that are required for the successful maturation of oocytes and fertilization. The absence of cumulus cells impairs embryo production. Denuded oocytes in culture cannot undergo normal fertilization and development. The structural integrity of the cumulus cell extracellular matrix (ECM) is
e.g., heavy chain of inter-alpha-trypsin inhibitor (ITIH) [60], pentraxin-3 (PTX3) [61,62], and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) [1], are required for maintaining cumulus integrity, thus ensuring cumulus expansion and oocyte maturation [2,61,63].
Oocyte maturation refers to the progression of the oocyte nucleus from the germinal vesicle to the metaphase II stage. Nuclear maturation involves GVBD, condensation of chromosomes, metaphase I spindle formation, separation of the homologous chromosomes with extrusion of the first polar body and arrest at metaphase II [64]. During oocyte maturation, cumulus cells change from a compact cell into a dispersed structure of cells for the synthesis of extracellular matrix, spaces between cumulus cells in cumulus–oocyte complexes become enlarged, and cells become embedded in a sticky, mucified matrix, This phenomenon is referred to as cumulus expansion. Cumulus expansion is thought to influence a variety of fundamental developmental changes during oocyte maturation. Expansion of the cumulus-oocyte complex correlates with the outcome of oocyte maturation, fertilization, and embryo development. Therefore, detailed functional studies of cumulus expansion seem to be required to elucidate the mechanism of oocyte maturation. Cumulus expansion involves hyaluronan accumulation in the intercellular spaces of cumulus cells, and its induction by gonadotropins is crucial for oocyte maturation [3]. Oocyte-secreted molecules, e.g.,
growth differentiation factor 9 and bone morphogenetic protein 15, also affect cumulus expansion [65,66].
Thus, bidirectional intercellular communication between oocytes and their surrounding cumulus cells is important for the development of an egg that is competent to undergo fertilization and embryogenesis [3,67,68]. In chapter 3, I will describe our findings concerning the importance of cumulus cell expressing SERPINE2 in oocyte maturation.
Chapter 2
Study I: SERPINE2, a Serine Protease Inhibitor Extensively Expressed in Adult Male Mouse Reproductive Tissues, May Serve as a Murine Sperm Decapacitation Factor
2.1 Introduction
SERPINE2 protein is widely expressed in various tissues, whereas the highest level is found in the seminal vesicles [69]. The Serpine2 gene knockout causes abnormal alterations in SVS components, which may result in an imbalance between PLAU, expressed in seminal vesicles [70], and SERPINE2. Thus, the vaginal plug becomes soft and shortened after mating and eventually impairs male fertility [71].
However, Pang et al. [17]found that removal of the coagulating glands, resulting in the absence of a vaginal plug, did not seem to explain the reduced fertility, which indicates that seminal vesicles may contribute certain factors to modulate fertility. Therefore, in addition to having a role in balancing the activity of proteases, SERPINE2 may also play a role in male reproduction. Although SERPINE2 was detected in portions of male reproductive tissues [45,69] the comprehensive expression patterns and functions of
Proteins from the seminal plasma interact with sperm by modifying the sperm surface membrane, an essential process in maintaining sperm viability, thus modulating their functions [20]. Decapacitation factors are one of those proteins in seminal plasma important for preventing premature capacitation. The sperm capacitation and acrosome reaction are essential for sperm-egg fertilization but premature capacitation will shorten sperm’s lifespan and potentially miss the fertilization opportunity. In the study I, we purified SERPINE2 protein from the mouse seminal vesicle to delineate its protein distribution in the male reproductive system and to study its effects on sperm function.
2.2 Materials and Methods
Animals
Specific pathogen-free outbred ICR mice were purchased from BioLASCO Taiwan (Taipei, Taiwan). The animals were bred based on the technology derived from Charles River Laboratories (Wilmington, MA) and were maintained in the Animal Center at the Department of Medical Research, Mackay Memorial Hospital, and were treated according to institutional guidelines for the care and use of experimental animals.
They were housed under controlled lighting (12L:12D) at 21–22ºC and provided with water and NIH-31 laboratory chow ad libitum.
Protein Purification and Analysis
Adult male mice (10–12 weeks old) were killed by cervical dislocation. The SVS was successively collected, centrifuged, and fractionated by ion-exchange chromatography using a diethylaminoethyl Sephacel (GE Healthcare Life Sciences, Piscataway, NJ) column and gel filtration with a Sephadex G-75 (GE Healthcare Life Sciences) column as previously described [36]. The potential SERPINE2-containing peak II eluted from the Sephadex G-75 column was further subjected to a heparin Sepharose 6 Fast Flow (2.6- X 10-cm) column (GE Healthcare Life Sciences) preequilibrated with 0.1 M Tris-HCl, 0.01 M sodium citrate, and 0.225 M NaCl (pH 7.4). After the nonretarded fractions were washed out, the column was eluted with 0.5 M NaCl in 0.1 M Tris-HCl and 0.01 M sodium citrate at pH 7.4 at a flow rate of 0.5 ml/min; fractions (2 ml) were collected, and absorbance records are shown in Figure 1A.
The protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The N-glycoconjugate was removed from a glycoprotein using a PNGase F kit (New England Biolabs, Beverly, MA) following the manufacturer’s instructions.
Protein Identification by Mass Spectrometry
Purified protein was resolved by SDS-PAGE on a 10% slab gel. Protein bands on the SDS gel were excised and subjected to in-gel digestion with trypsin. In brief, the gel was washed in a solution of 50% (v/v) acetonitrile and 100 mM NH4HCO3 and digested by trypsin overnight at 37 ºC. The tryptic peptides were then extracted with a solution of 60% (v/v) acetonitrile and 1% (v/v) trifluoroacetic acid (TFA), lyophilized, resuspended in 0.1% (v/v) TFA, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) equipped with an 1100 series HPLC unit (Agilent Technologies, Palo Alto, CA) and an LTQ FT hybrid mass spectrometer (Thermo Electron, San Jose, CA). MS/MS data were used for protein identification, using MASCOT search engine software (http://www.matrixscience.com), based on the International Protein Index databases (http://www.ebi.ac.uk/IPI).
Activity Assay
The inhibitory activity of SERPINE2 toward PLAU (also named uPA) was assayed using a uPA colorimetric assay kit (Millipore, Billerica, MA) according to the manufacturer’s protocol. In brief, 5 µg of purified SERPINE2 protein was incubated with 5 units of PLAU for 1 h at 37ºC. Subsequently, assay buffer and chromogenic substrate were added and incubated for 30 min at 37°C. The absorbance was read at 405 nm.
Antibody Production and Usage
Antisera against SERPINE2 were produced using New Zealand white rabbits.
Purified SERPINE2 protein in normal saline (0.4 mg/ml) was emulsified with Freund’s complete adjuvant (1:1, v/v). In total, 2 ml of the mixture was subcutaneously injected in multiple sites in individual rabbits. Two rabbits were boosted twice every 3 weeks with the mixture of the same amount of purified protein and Freund’s incomplete adjuvant (1:1, v/v). Antisera were collected 10 days after the last injection. Purified SERPINE2 protein (200 µg) was conjugated to AminoLink beads (Pierce) according to the manufacturer’s instructions. Antisera against SERPINE2 were adsorbed by the conjugated beads to remove the specific antibody against SERPINE2. The treated antiserum was used as the control antiserum. Antisera were also used to develop a custom-made sandwich-style ELISA kit by Taiwan Advanced Bio-Pharm (Taipei, Taiwan). The kit was used to estimate the SERPINE2 protein concentration in SVS, which was collected separately from 6 male mice at the age of 12 weeks.
Western Blotting
Proteins were resolved using SDS-PAGE on a 10% slab gel (8.2 × 7.3 × 0.075 cm) and stained with Coomassie Brilliant blue or transferred to a nitrocellulose
membrane for immunostaining. Membranes were blocked with 10% (w/v) skim milk in PBS (blocking solution) for 2 h and then incubated with anti-SERPINE2 antiserum or control antiserum (1:8000 dilution) in blocking solution for 1 h at room temperature.
After gentle agitation over four changes of PBS for 10 min each, membranes were immunoreacted with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (GE Healthcare Life Sciences) diluted to 1:10 000 in blocking solution for 1 h. Immunoreactive bands were revealed using an enhanced chemiluminescence substrate according to the manufacturer’s instructions (Pierce).
Immunohistochemical Staining of the Accessory Gland of Male Mice
Murine (~12 weeks old) reproductive tissues were collected, fixed in formalin, embedded in paraffin, and cut into 5-lm sections. After the slides were deparaffinized and hydrated, they were placed in a plastic slide holder filled with antigen retrieval AR-10 solution (BioGenex, San Ramon, CA), soaked in a 70°C water bath, rapidly boiled to 95°C, and maintained for 15 min. While cooling to room temperature for 30 min, the slides were treated with 3% (v/v) H2O2 in PBS for 15 min, blocked with 10%
(v/v) normal goat serum in PBS (blocking solution) for 1 h at room temperature, and then incubated with anti-SERPINE2 antiserum or the control antiserum diluted 1:1000 in the blocking solution at 4 ºC for 16 h. After slides were washed, they were treated
with biotin-conjugated goat anti-rabbit IgG (~3 µg/ml) (Zymed Laboratories, South San Francisco, CA) in blocking solution for 1 h at room temperature. Slides were washed again and then incubated with HRP-conjugated streptavidin (~1 µg/ml) (Zymed Laboratories) in blocking solution for 40 min at room temperature. Protein signals were detected by 3-amino-9-ethylcarbazole staining (Zymed Laboratories). Slides were then counterstained with hematoxylin (Vector Laboratories, Burlingame, CA) and photographed using an Olympus BX 40 microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP-70 digital camera.
Preparation of Spermatozoa
Epididymides and testes were immediately removed after male mice (;12 weeks old) were killed. Caudal epididymides were slit in prewarmed Biggers, Whitten, and Whittingham (BWW) medium and incubated at 37 ºC in 5% CO 2 for 15 min to allow motile sperm to swim upward. Motile sperm in the upper layer were collected. The caput and corpus regions of epididymides were treated in the same method as described above but were gently filtered through a 70 µm nylon cell strainer (BD Falcon, Franklin Lakes, NJ) to remove debris. Testes were decapsulated by cutting the tunica albuginea to expose seminiferous tubules. An 18-gauge needle was used to aspirate the seminiferous tubules and push them through. The dispersed seminiferous tubules were
cut into pieces and filtered through a 70-lm nylon cell strainer (BD Falcon) to collect the free seminiferous cells. To isolate ejaculated uterine and oviductal sperm, female mice (6 weeks old) were induced to superovulate by an intraperitoneal injection of 10 IU of equine chronic gonadotropin (China Chemical and Pharmaceutical, Hsinchu, Taiwan), followed by an intraperitoneal injection of 10 IU of human chorionic gonadotropin (China Chemical and Pharmaceutical) 48 h later, and were subsequently mated with male mice (~16 weeks old). Female mice with plugged vaginas were killed, and the ejaculated sperm in the uterine cavity were collected within 1 h. In brief, the semen filtered through a 70 µm nylon cell strainer (BD Falcon) was repeatedly agitated by pipetting with PBS. The sperm solution was washed by centrifuging it three times at 100 X g for 10 min. Sperm were then fixed using 4% (w/v) paraformaldehyde in an Eppendorf tube for 20 min at room temperature, transferred onto slides, and allowed to dry. Oviductal sperm were collected the next day after mating by flushing the oviduct with PBS. Sperm were transferred using a mouth pipette onto slides and fixed in 4%
(w/v) paraformaldehyde for subsequent immunostaining analysis.
Immunolocalization of SERPINE2 on Spermatozoa
To determine whether SERPINE2 protein is originally a sperm-binding protein, freshly prepared epididymal and testicular spermatozoa were fixed using 4% (w/v)
paraformaldehyde and allowed to dry on a glass slide and washed twice with PBS. After slides were incubated in blocking solution of PBS containing 10% (v/v) normal goat serum for 1 h at room temperature, they were incubated with anti-SERPINE2 antiserum or control antiserum at a dilution of 1:100 in blocking solution for 1 h. The slides were washed three times with PBS to remove excess antibodies before they were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Vector Laboratories) diluted 1:500 in blocking solution for 40 min. All slides were then washed with PBS and counterstained with 5μg/ml Hoechst 33258 stain. After three brief rinses with PBS, the slides were mounted in 100μl of ProLong Gold antifade medium (Invitrogen Molecular Probes, Eugene, OR) and photographed using an epifluorescence microscope (Olympus BX 40) equipped with an Olympus DP-70 digital camera. To determine whether exogenous SERPINE2 protein can bind to epididymal sperm, 0.5 μM SERPINE2 was incubated with living sperm in Eppendorf tubes for 20 min at 37℃.
Unbound SERPINE2 protein was washed away by centrifuging the sperm in the PBS solution at 100 x g for 5 min at room temperature. Sperm were then fixed using 4%
(w/v) paraformaldehyde in an Eppendorf tube for 20 min at room temperature, transferred onto slides, and allowed to dry. Immunostaining was done as described above, except a dilution of 1:1000 was used for anti-SERPINE2 antiserum and control antiserum. The same dilution was also used to examine SERPINE2 on ejaculated and
oviductal sperm, without incubation with the exogenous SERPINE2 protein. To examine the correlation between SERPINE2-bound sperm and sperm capacitation, oviductal sperm were double fluorescence labeled by using indirect immunofluorescence and chlortetracycline (CTC) fluorescence staining, an empirical method used to morphologically assess sperm capacitation [72,73]. In brief, sperm
oviductal sperm, without incubation with the exogenous SERPINE2 protein. To examine the correlation between SERPINE2-bound sperm and sperm capacitation, oviductal sperm were double fluorescence labeled by using indirect immunofluorescence and chlortetracycline (CTC) fluorescence staining, an empirical method used to morphologically assess sperm capacitation [72,73]. In brief, sperm