During ovulation, SERPINE2 and PLAU expression is coordinated in mice [46], whereas SERPINE1 and PLAT expression is coordinated in monkeys and rats [90-92].
This indicates that the PA system has species-specific expression patterns in the ovary.
PA expression levels are upregulated in cumulus cells just before ovulation [93] and are involved in follicle wall rupture during ovulation [38,47,48,69]. PAs and their cognate serpin inhibitors have been detected in cumulus cells [46]; however, their involvement in oocyte maturation during pre-ovulation needs clarification. Several studies have reported cumulus expansion is essential for oocyte maturation. Many cumulus proteins are required for regulating cumulus structure and cumulus expansion, such as
Hyaluronan synthase 2 (Has2), PTX3, Versican (Vcan) and Tnfaip6. In study II, higher
SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. Therefore, we here assumed that highSERPINE2 levels were correlated with cumulus expansion and oocyte immaturity. To verify this, we used mouse cumulus–oocyte complexes (COCs) as a model for evaluating the association of SERPINE2 levels with cumulus expansion and subsequent oocyte maturation.
3.2 Materials and Methods
Ethics statement
This study was approved by the Mackay Memorial Hospital Institutional Review Board (reference number 09MMHIS024) with written consent for the use of human cumulus cells. Written consent for the use of cumulus cells was obtained from 20 patients undergoing intracytoplasmic sperm injection treatment. All animals contributed to this study were maintained in the Animal Center at the Department of Medical Research, Mackay Memorial Hospital. The animal use protocol has been reviewed and approved by the Mackay Memorial Hospital Institutional Animal Care and Use Committee with an approval number MMH-A-S-100-45. All efforts were made to minimize suffering.
Collection of human cumulus cells
Reproductive Medicine, Mackay Memorial Hospital, Taiwan received controlled ovarian hyperstimulation by application of the gonadotropin-releasing hormone antagonist protocol. COCs from follicles >14 mm were collected using transvaginal ultrasound and a 16-gauge needle and were exposed to 80 IU hyaluronidase in Quinn's Advantage Fertilization medium (Sage BioPharma, Bedminster, NJ) for 20 s at 37°C to dissolve hyaluronan. Of the 46 COCs, 26 and 20 had mature and immature oocytes, respectively. The cumulus cells were individually separated from the COCs under an Olympus SZX7 stereomicroscope (Tokyo, Japan). They were mixed with 20 µl of extraction buffer from the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Foster City, CA) for total RNA isolation and stored at −80°C until use. Cumulus cells individually collected from 10 other COCs were fixed on slides using 4% (v/v) paraformaldehyde for immunohistochemical staining.
Collection of mouse cumulus cells
The mice (age, 21–24 days) were injected with 5 IU of pregnant mare serum gonadotropin (PMSG; Sigma-Aldrich, St. Louis, MO) and sacrificed by cervical dislocation after 46 h. The ovaries were removed and briefly rinsed with PBS. COCs were isolated by puncturing antral follicles with a 30-gauge needle under an Olympus SZX7 stereomicroscope. To study the effect of luteinizing hormone on Serpine2 and
Plau expression in cumulus cells during oocyte maturation, PMSG-primed mice were
injected with 5 IU of human chorionic gonadotropin (hCG; Sigma-Aldrich). Ovaries were removed 3, 6, and 9 h after hCG treatment. COCs were isolated by puncturing antral follicles as described above. For post-ovulation COCs, the ovaries were removed 12 h after hCG injection, and the COCs were collected by flushing the oviducts with PBS. All COCs were treated with 150 IU hyaluronidase in PBS for 3 min at 37°C, the oocytes were removed, and cumulus cells were collected by centrifugation at 1000 ×g for 3 min at room temperature.
Quantitative real-time RT-PCR (qRT-PCR)
Total RNA of cumulus cells was extracted using the Arcturus PicoPure RNA Isolation Kit and directly reverse transcribed into a 50 µl first-strand cDNA pool using a High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR was performed using primers (Table 1) [42]. The housekeeping genes, human ribosomal protein L19 and mouse hypoxanthine guanine phosphoribosyltransferase gene, were used as internal loading controls to normalize relative gene expression levels. PCR amplification efficiency for each tested gene was examined to ensure that it was equivalent to that of the housekeeping gene examined in a cDNA dilution series. PCR was performed in a total volume of 20 µl, containing 50 ng
of tissue cDNA, 250 nM each of the forward and reverse primers, and 10 µl of 2×
SYBR Green Master Mix (Applied Biosystems). All reactions were performed in triplicate and run on an ABI/PRISM 7500 Fast Sequence Detection System (Applied Biosystems) under the following conditions: 95°C for 20 s, and then 40 cycles at 95°C for 3 s and 60°C for 30 s. The threshold cycle (Ct) was defined as the fractional cycle number at which the reporter fluorescence, i.e., the number of amplified copies, reached a fixed threshold. Melting curve analysis was performed to verify that only a single product had formed in the reaction. The identity of the PCR products was confirmed by DNA sequencing. Relative quantification of mRNA expression was calculated by the 2−ΔΔCt method [90].
SERPINE2 proteins and anti-SERPINE2 antiserum
SERPINE2 proteins and anti-SERPINE2 antiserum were prepared (Chapter 1).
To prepare control antiserum, anti-SERPINE2 antiserum was adsorbed onto SERPINE2-conjugated beads for removing the specific anti-SERPINE2 antibody (Chapter 1).
SERPINE2 and PLAU immunolocalization and hyaluronan status on COCs
COCs were transferred onto slides, air dried, and fixed in 4% paraformaldehyde
for 15 min. The slides were incubated in blocking solution [10% (v/v) normal goat serum in PBS for 1 h at room temperature and then incubated with anti-SERPINE2 or control antiserum (1:1000), with rabbit anti-PLAU antiserum (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or with pre-immune rabbit serum (1:500; Jackson ImmunoResearch, West Grove, PA) in blocking solution at 4°C for 16 h. To assess the hyaluronan status in cumulus cells, slides were incubated with biotinylated hyaluronic acid binding protein (HABP; 1:200, Sigma-Aldrich, cat. no. H9910) in blocking solution at 4°C for 4 h. After washing three times in PBS with slight agitation for 5 min each, the slides were treated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:1000; Jackson ImmunoResearch) or with tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit IgG (1:1000; Jackson ImmunoResearch) in blocking solution for 1 h at room temperature or with streptavidin-conjugated Alexa Fluor 488 (1:1000; Jackson ImmunoResearch) in blocking solution for 2 h at room temperature. The slides were washed again and then counterstained with 5 µg/ml Hoechst 33258. After three brief rinses with PBS, the slides were mounted in 100 µl 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.
In vitro maturation (IVM)
To assess the extent of cumulus cell expansion, COCs isolated from PMSG-primed ovaries that had even diameters of approximately 100 µm and contained a nucleus (germinal vesicle, GV) were cultured in IVM medium as described previously [94,95]
with some modifications. The IVM medium consisted of MEMα medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 0.23 mM sodium pyruvate, 75 mU/ml follicle-stimulating hormone (FSH), 50 mg/l streptomycin, 60 mg/l penicillin, and 1 μg/l epidermal growth factor (EGF), pH 7.4. COCs were incubated in 150-μl microdrops of IVM medium supplemented with SERPINE2 (0.03, 0.06, or 0.12 mg/ml), anti-SERPINE2 antibody (1:1000), amiloride (300 μM), or PLAU (20 U; Millipore, Billerica, MA) and overlaid with mineral oil for approximately 16–20 h in a humidified 5% CO2 atmosphere at 37°C.
For control experiments, COCs were incubated in IVM medium without supplementation. After IVM, the diameters of expanded cumulus cells were scored.
Next, the COCs were treated with 150 IU hyaluronidase in IVM medium for 3 min at 37°C, and cumulus cells were removed by repeated pipetting. The morphology of oocyte nuclei was observed, and the oocytes were classified as immature GV or metaphase I (MI) stage or mature (MII stage, with the extrusion of the first polar body).
Oocyte maturation rate was determined after 16 h of culture by counting the number of
MII oocytes among the total oocytes used in an assay.
Treatment of COCs with small interfering RNA (siRNA)
siRNA against mouse Serpine2 (catalog #20720-Serpine2; Dharmacon, Thermo Fisher Scientific, Lafayette, CO) and a non-targeting negative control siRNA (catalog
#D-001206-05; Dharmacon, Thermo Fisher Scientific) dissolved in Accell siRNA delivery media were used according to the manufacturer's instructions. COCs isolated from PMSG-primed ovaries were incubated with 1, 2, or 3 μM siRNAs for 24 h in 150 µl MEMα medium supplemented with 10 µM milrinone (a phosphodiesterase inhibitor, Sigma-Aldrich, cat. no. M4659), 50 mg/l streptomycin, 60 mg/l penicillin, 0.23 mM pyruvate, and 3 mg/ml bovine serum albumin (Sigma-Aldrich). The optimal concentration for both siRNAs was 3 μM. After 24 h incubation, the COCs were transferred to IVM medium and cultured in a humidified 5% CO2 atmosphere at 37°C for 16 h. Cumulus expansion and oocyte maturation were then assessed as described above. Serpine2 mRNA levels in cumulus cells were examined by qRT-PCR. To analyze whether SERPINE2 protein was knocked down, COCs were transferred onto slides and examined by immunohistochemistry as described above.
Construction of the mouse Serpine2 expression vector
The DNA fragment of the pIRES2-DsRed2 vector (Clontech Laboratories, Mountain View, CA) containing the multiple cloning site (MCS) and the red fluorescence protein coding region (DsRed2) was amplified by PCR using primer pairs bearing EcoRI sites (forward primer 5′-TTCGAATTCTGCAGTCGACGGTACC-3′, reverse primer 5′-TTTGAATTCATCTAGAGTCGCGGCCGC-3′; Fig 15a). Thirty-five PCR cycles were performed under the following conditions: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The PCR product was verified by agarose gel electrophoresis and DNA sequencing and ligated into an
EcoRI-digested pCX-EGFP vector (Addgene, Cambridge, MA) to form the
pCX-DsRed2 intermediate vector (Fig. 15b and c). Total RNA was extracted from mouse seminal vesicles using an RNeasy Mini Kit (Qiagen, Valencia, CA) and reverse transcribed into cDNA with a High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer’s instructions. The 1220-bp full-length mouse Serpine2 cDNA (NCBI Reference Sequence NM_009255.4) was amplified by RT-PCR from the cDNA pool using a Serpine2 primer pair (forward primer
5′-GAAGGAACCATGAATTGGC-3′, reverse primer 5′-TTCCTTTGTCTGTCCTTCAGG-3′). Thirty-five cycles of PCR were performed
under the following conditions: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The PCR product was verified by agarose gel
electrophoresis and DNA sequencing and cloned into the pGEM-T Easy vector (Promega, Madison, WI) by TA cloning. The full-length Serpine2 cDNA was excised with XmaI and cloned into MCS of the pCX-DsRed2 vector to create the Serpine2 expression vector pCX-Serpine2-DsRed2 (Fig. 15d). The construct was sequenced to verify the sequence and orientation of the reading frame. This construct enabled the simultaneously translation of both SERPINE2 and DsRed2 for monitoring SERPINE2 protein expression by red fluorescence.
Serpine2 overexpression in COCs
COCs isolated from PMSG-primed ovaries were transfected with 500 ng of the
Serpine2 expression vector pCX-Serpine2-DsRed2 or the vehicle vector pCX-DsRed2
using PolyJet DNA In Vitro Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD) in 150 µl of MEMα medium supplemented with 10 µM milrinone as mentioned above but without FSH and EGF, for 12 h. The COCs were washed three times using IVM medium, transferred to fresh medium, and cultured in a humidified 5% CO2
atmosphere at 37°C for 16 h. Cumulus expansion and oocyte maturation were assessed as described above. Serpine2 mRNA levels in cumulus cells were examined by qRT-PCR.
Statistical analysis
Data are presented as means ± SD. Differences were analyzed by one-way analysis of variance followed by the Bonferroni post hoc test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.
3.3 Results
SERPINE2 was highly expressed in cumulus cells of immature human oocytes
We analyzed the expression levels of the four SERPINs of the PA system in cumulus cells of the mature human oocyte by qRT-PCR and found that SERPINE2 were the most highly expressed (Fig. 9A). Next, we compared SERPINE2 mRNA expression levels in cumulus cells collected from mature and immature human oocytes. Cumulus cells from immature oocytes expressed significantly (P < 0.0001) higher SERPINE2 mRNA levels than those from mature oocytes (Fig. 9B). Similarly, considerably more SERPINE2 protein was detected in cumulus cells from immature human oocytes at the GV and MI stages (Fig. 9C, a and b, respectively) than in those from mature MII oocytes or with the control staining of MII oocytes (Fig. 9C, c and d, respectively).
Other similar cases are shown in Fig. 16.
Serpine2 and Plau were highly expressed in mouse cumulus cells during oocyte
maturation
We analyzed the expression profiles of the four Serpins of the PA system in cumulus cells surrounding mature mouse oocytes. Similar to the results with human cumulus cells, Serpine2 mRNA was the most highly expressed in mouse cumulus cells (Fig. 10A). Next, we analyzed the gene expression patterns of SERPINE2-targeted serine proteases in the cumulus cells of mature mouse oocytes using qRT-PCR. Plau mRNA was the most highly expressed, followed by Plat and Prss8 mRNAs. F2 mRNA was almost undetectable (Fig. 10B). To examine the in vivo expression pattern of Serpine2 and Plau mRNAs in mouse cumulus cells during oocyte maturation, the cumulus cells were collected at various intervals during gonadotropin-induced oocyte maturation. Serpine2 mRNA was highly expressed 46 h after PMSG treatment and reached a maximum level 3 h after hCG administration, gradually decreasing to its lowest level 12 h after hCG administration (Fig. 10C). Plau mRNA was at a low level 46 h after PMSG treatment; however, it peaked 3 h and 6 h after hCG treatment and then gradually decreased to a low level after 12 h (Fig. 10D). The relative changes in
Plau mRNA levels were much greater than the changes in Serpine2 mRNA levels (Fig.
10C and 10D). Expression of SERPINE2 and PLAU proteins was consistent with their mRNA expression in cumulus cells. SERPINE2 was at a relatively high level following
PMSG administration and 3 h after hCG treatment (Fig. 10E, a and b). PLAU was at a relatively low level after PMSG treatment but peaked approximately 3–6 h after hCG treatment (Fig. 10F, a–c). From 6 h after hCG on, SERPINE2 protein levels were gradually decreased to a very lower level (Fig. 10E, c–f); on the contrary, PLAU protein levels were still at higher levels at 6 h after hCG (Fig. 10F, c) and then sharply decreased to a very low level thereafter (Fig. 10F, d–f).
Serpine2 silencing or SERPINE2 protein blockage had no effect on cumulus
expansion and oocyte maturation in vitro
IVM is often used to culture compact immature oocytes collected from PMSG-primed ovaries (Fig. 11A, a) for developing MII mature oocytes with fully expanded cumulus cells (Fig. 11A, b). To examine the effect of Serpine2 silencing on oocyte maturation in cumulus cells, siRNA was used to knockdown Serpine2 mRNA expression during IVM. Cells were also treated with SERPINE2 antiserum to examine the effect of blocking SERPINE2 protein. No detrimental effects on COC structure or morphology (Fig. 11A, c–e) or on the extent of cumulus expansion (Fig. 11B) were observed, although Serpine2 mRNA was significantly decreased (P < 0.0001) and SERPINE2 protein was knocked down by the introduction of Serpine2 siRNA (Fig. 11C and Fig. 17, respectively). The treatments had no effect on oocyte maturation (Table 2).
As shown in Fig 11D, more than 70% of oocytes reached the MII stage even when Serpine2 mRNA in cumulus cells was knocked down. Oocyte maturation was comparable in the media with and without control or Serpine2 siRNAs or specific anti-SERPINE2 antiserum. Taken together, these findings indicated that eliminating SERPINE2 in cumulus cells had no apparent effect on cumulus expansion and oocyte maturation.
SERPINE2 overexpression in cumulus cells impaired cumulus expansion and
oocyte maturation in vitro
To test whether high SERPINE2 levels affected cumulus expansion and oocyte maturation, mouse COCs were transfected with a vector carrying Serpine2. The COC morphology was symmetrical with the outward expansion pattern of cumulus cells from the oocyte in both the untreated control and after transfection with control plasmid DNA (Fig. 12, a and b); however, the cumulus cell was compact or in an unexpanded state after transfection with the Serpine2 plasmid (Fig. 12A, c). SERPINE2 protein was significantly overexpressed in cumulus cells after transfection and culturing for 16 h, although most of the protein expression was in the outer layer of cumulus cells (Fig.
12A, d). Similarly, exogenously added SERPINE2 resulted in compact, unexpanded cumulus cells that tightly encircled the oocyte (Fig. 12A, e–g). Serpine2 mRNA was
significantly overexpressed in cumulus cells after transfection and culturing for 16 h (Fig. 12B). Serpine2 overexpression significantly reduced the extent of cumulus expansion compared to transfection with the control plasmid (P < 0.0001; Fig. 12C, gray bars). Exogenous SERPINE2 also significantly inhibited cumulus cell expansion compared to the control (Fig. 12C, black bars). Serpine2 overexpression in cumulus cells or exogenously added SERPINE2 significantly reduced oocyte maturation, with most oocytes halting at the MI stage (Table 3). Introduction of the Serpine2 plasmid into cumulus cells significantly reduced oocyte maturation by approximately 45% compared with the control group (Fig. 12D, gray bars). SERPINE2 supplemented exogenously also significantly reduced oocyte maturation by approximately 26–42% (Fig. 12D, black bars).
PLAU protein was involved in cumulus expansion and oocyte maturation
Since PLAU was the most highly expressed serine protease in cumulus cells, we examined PLAU effects on cumulus expansion and oocyte maturation. COC expansion was visible at 6 h of culture (Fig. 13A, a) and had fully expanded cumuli with an average diameter of 236 µm after approximately 16–20 h of culture (Fig. 13A, b, and 13B, open bar). PLAU supplementation significantly expanded the COCs to an average diameter of 291 µm (Fig. 13A, d, and 13B, gray bar), and the expansion occurred earlier
at 6 h during IVM compared with that in the control group (Fig. 13A, a and c).
Furthermore, the PLAU-supplemented cumulus cells degraded earlier (at 20 h) than the control cells (Fig. 13A, b and e), which generally degraded at 24 h. To determine whether the PLAU effect on oocyte maturation was specific, amiloride, a specific inhibitor of PLAU, was added to the IVM medium. As shown in Fig. 13A, f, and 5B (black bar), cumulus expansion was significantly diminished, and cumulus cells remained encircling the GV oocyte at 20 h, similar to the effects of SERPINE2 addition (Fig. 13A, g, and 4C, black bars). To further demonstrate that the inhibition of cumulus expansion was due to PLAU suppression, amiloride or SERPINE2 was coincubated with PLAU during IVM. Intriguingly, the COC morphology appeared normal (Fig. 13A, g and h), and the extent of cumulus cell expansion was comparable to that in the control group (Fig. 13B, hatched bars). PLAU significantly promoted oocyte maturation (P <
0.05), whereas amiloride significantly reduced oocyte maturation (P < 0.0001) (Fig.
13C and Table 4). Coincubation with PLAU and amiloride or SERPINE2 (Fig. 13C, hatched bars) reduced maturation to levels comparable with the control group (approximately 53% and 56%, respectively, vs. 67% for the control). Taken together, these data suggested that PLAU was involved in cumulus expansion and oocyte maturation and that its effects could be modulated by SERPINE2.
Excessive PLAU and SERPINE2 altered matrix gene expression and the
hyaluronan status of cumulus cells during IVM
To examine the effect of excessive PLAU and SERPINE2 on the temporal gene expression pattern of the matrix genes in cumulus cells, cumulus cells at 3, 6, and 16 h, the critical time points, during IVM culture were collected from COCs and analyzed by qRT-PCR. PLAU significantly enhanced but SERPINE2 significantly down-regulated cumulus hyaluronan synthase 2 (Has2) mRNA levels at 3 and 6 h of IVM compared with that in the control IVM group (Fig. 14A, a). Vcan mRNA levels in cumulus cells were significantly diminished by exogenous SERPINE2 at 3 and 6 h and by PLAU at 6 h of IVM, but with a PLAU-induced surge at 16 h of IVM (Fig. 14A, b). Cumulus
Tnfaip6 mRNA levels were enhanced by exogenous PLAU after 3 h of IVM; however,
this enhancement disappeared at 6 and 16 h of IVM culture. SERPINE2 showed no effect on Tnfaip6 mRNA expression at all the time points (Fig. 14A, c). Furthermore, exogenous PLAU and SERPINE2 had no effect on Ptx3 mRNA expression in cumulus cells (Fig. 14A, d). HAS2 is a critical enzyme required for matrix hyaluronan synthesis (63). Since Has2 mRNA expression is the most affected, we analyzed the hyaluronan status of cumulus cells during IVM. After 6 h of culturing, mouse COCs showed moderate expansion with relatively high hyaluronan contents around the cumulus matrix (Fig. 14B, a and e), in contrast to the negative staining control (Fig. 14B, b and f).
Hyaluronan staining looked over the cell; however, the staining was clearly outside of the cumulus cell when ovarian tissue slides were stained. Thus, this staining pattern is probably caused by the steric stacking of cumulus cells (Fig. 18). Intriguingly, exogenous PLAU increased hyaluronan contents (Fig. 14B, c and g), while SERPINE2 supplementation decreased the contents on the cumulus matrix compared with that in the control group (Fig. 14B, d and h).
3.4 Discussion
PLAU and SERPINE2 were the most abundant PA and PA inhibitor, respectively, in murine cumulus cells, and their gene expression levels were coordinately regulated during gonadotropin-induced oocyte maturation (Fig. 10). SERPINE2 decreased rapidly 6 h after hCG administration, consistent with apparent cumulus expansion, whereas PLAU remained at a high level. Thus, the net proteolytic activity of PLAU may contribute to the initiation of cumulus expansion. This interplay appears to suggest that the net activity of PLAU may be crucial for cumulus expansion and subsequent oocyte maturation. Furthermore, we found that PLAU depletion via its specific inhibitor, amiloride, largely impaired these biological processes (Fig. 12). Hagglund et al.
reported high Serpine2 mRNA levels and low Plau mRNA levels in mouse cumulus
cells [46] and suggested that SERPINE2 may provide inhibitory activity for protecting the mucified COC matrix from proteolytic degradation. However, we found that cumulus cells expressed both Plau and Serpine2 mRNA and protein during gonadotropin-induced oocyte maturation (Fig 10). Furthermore, in vivo, the granulosa cells, which are far more numerous, may also produce these proteins. Hence, we examined their relative expression in cumulus and granulosa cells after hCG 3, 6, and 9 h by immunohistochemistry. Granulosa cells expressed these proteins at levels similar to cumulus cells, especially at 3 and 6 h of hCG treatment (Fig. 19). PLAU and SERPINE2 detected in mural granulosa cells may also become associated with the COC matrix during cumulus expansion. We compared cumulus SERPINE2 and PLAU levels in COCs treated with hCG in vivo or cultured in vitro. Cumulus SERPINE2 levels had
cells [46] and suggested that SERPINE2 may provide inhibitory activity for protecting the mucified COC matrix from proteolytic degradation. However, we found that cumulus cells expressed both Plau and Serpine2 mRNA and protein during gonadotropin-induced oocyte maturation (Fig 10). Furthermore, in vivo, the granulosa cells, which are far more numerous, may also produce these proteins. Hence, we examined their relative expression in cumulus and granulosa cells after hCG 3, 6, and 9 h by immunohistochemistry. Granulosa cells expressed these proteins at levels similar to cumulus cells, especially at 3 and 6 h of hCG treatment (Fig. 19). PLAU and SERPINE2 detected in mural granulosa cells may also become associated with the COC matrix during cumulus expansion. We compared cumulus SERPINE2 and PLAU levels in COCs treated with hCG in vivo or cultured in vitro. Cumulus SERPINE2 levels had