Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish

Abstract

The progestin receptor membrane components (Pgrmcs) contain two paralogs, Pgrmc1 and Pgrmc2. Our previous research into single knockout of Pgrmc1 or Pgrmc2 suggests that Pgrmc1 and Pgrmc2 regulate membrane progestin receptor or steroid synthesis and therefore female fertility in zebrafish. Additional roles of Pgrmcs may not be determined in using single Pgrmc knockouts due to compensatory roles between Pgrmc1 and Pgrmc2. To address this question, we crossed single knockout pgrmc1 (pgrmc1^−/−) with pgrmc2 (pgrmc2^−/−), and generated double knockouts for both pgrmc1 and pgrmc2 (pgrmc1/2^−/−) in a vertebrate model, zebrafish. In addition to the delayed oocyte maturation and reduced female fertility, significant reduced ovulation was found in double knockout (pgrmc1/2^−/−) in vivo, though not detected in either single knockout of Pgrmc (pgrmc1^−/− or pgrmc2^−/−). We also found significant down regulation of nuclear progestin receptor (Pgr) protein expression only in pgrmc1/2^−/−, which was most likely the cause of reduced ovulation. Lower protein expression of Pgr also resulted in reduced expression of metalloproteinase in pgrmc1/2^−/−. With this study, we have provided new evidence for the physiological functions of Pgrmcs in the regulation of female fertility by regulation of ovulation, likely via regulation of Pgr, which affects regulation of metalloproteinase expression and oocyte ovulation.

Introduction

Ovulation is a physiological process where a mature, fertilizable oocyte is released from the surrounding follicular cells. The degradation in extracellular matrix of the oocytes is paramount for follicular rupture to occur. In vertebrates, ovulation is triggered by a luteinizing hormone (Lh) surge from the pituitary, which signals via the receptor (Lhcgr) in the granulosa cell. This initial signal then amplified via progestin synthesis in preovulatory follicles, which in turn activates nuclear progestin receptor (Pgr). The role of Pgr in ovulation is highly conserved across vertebrate species, as is evidenced by the inability of Pgr knockout female zebrafish, rats, and mice to ovulate (Kubota et al 2016, Lydon et al 1995, Zhu et al 2015). Further studies indicated that Pgr is an upstream regulator important for the increased expression of proteolytic enzymes and therefore promotes the rupture of follicles for releasing mature oocytes during ovulation (Liu et al., 2017, 2018).

As the downstream targets of Pgr, several proteolytic enzymes that may be responsible for follicle rupture during ovulation have been identified (Ogiwara et al 2005, Takahashi et al 2013). Several members of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family have been proposed to be important for ovulation. For example, MMPs and ADAMTS family were found to be induced in the ovarian follicles (Peluffo et al 2011, Piprek et al 2018, Rosewell et al 2015). In addition, inhibitors of MMPs using antibodies drastically suppressed in vitro ovulation in medaka (Ogiwara et al 2005). Pharmacologic inhibition of metalloproteases also indicates the importance of these proteinases in ovulation (Butler et al 1991, REICH et al 1985). However, since mice that lack metalloproteinases die either in utero or shortly after birth (Carmeliet et al 1993, Enomoto et al 2010, Holmbeck et al 1999, Kelly et al 2005, Peschon et al 1998, Vu & Werb 2000), the roles played by these proteases in ovulation remains unclear.

As in mammals, only two progestin receptor membrane component (Pgrmc) paralogs (Pgrmc1 and Pgrmc2) have been identified in zebrafish. They have been suggested as progestin receptors, or adaptor proteins, which facilitate other receptors to mediate progestin signaling (Peluso et al 2008, Thomas 2008, Zhu et al 2008). Pgrmcs take part in various physiological and biochemical processes that are important for normal reproduction. For instance, Pgrmc1 is regulated by progesterone and can also affect progesterone metabolism (Rohe et al 2009). In addition, Pgrmc1 may affect oocyte maturation via regulating expression and trafficking of mPRα to localize at cell surface (Thomas et al 2014, Wu et al 2018). Furthermore, Pgrmc1 has also been suggested to mediate antiapoptotic and antimitotic actions of progesterone in rat granulosa cells (Peluso et al 2006). Recent investigations in Pgrmc1 and Pgrmc2 knockout mice have suggested that Pgrmcs are required for normal fertility in females. Conditional ablation of Pgrmc1 results in reduced fertility in female mice, while knocking out Pgrmc2 causes premature reproductive senescence in female mice, possibly due to post-implantation failure (Clark et al 2016, McCallum et al 2016). Similar reduced fertility was also observed in pgrmc1^−/− and pgrmc2^−/− zebrafish (Wu et al 2019, Wu et al 2018). However, whether Pgrmc1 and Pgrmc2 compensate each other is still unknown. In the present study, we generated double knockouts for Pgrmc genes in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing technology, characterized phenotypes of these knockouts, and examined the molecular mechanisms underlying the actions of Pgrmc. We found reduced spawning frequencies and number of embryos in double knockouts pgrmc1/2^−/−. In addition, we observed reduced ovulation in vivo. Significant reduced Pgr protein expression in pgrmc1/2^−/− likely caused, at least partially, for attenuated ovulation in pgrmc1/2^−/−.

Materials and Methods

Zebrafish

The wildtype zebrafish (Danio rerio) strain used in this investigation, the Tübingen strain, was initially obtained from the Zebrafish International Resource Center, then propagated in our lab at East Carolina University. We used previously described methods to generate pgrmc1^−/− and pgrmc2^−/− single mutant lines (Wu et al 2019, Wu et al 2018). Double knockouts pgrmc1/2^−/− were generated by crossing of pgrmc1^−/− with pgrmc2^−/−. Fish were kept under a photoperiod of 14h light and 10h dark (lights on at 09:00, lights off at 23:00), with water temperature around 28.5°C, pH ~7.2, and salinity conductivity ranging from 700 to 1,200 μS in automatically controlled zebrafish rearing systems (Aquatic Habitats Z-Hab Duo systems, Florida, USA). Fish were fed twice a day and supplied with newly hatched brine shrimp. All the animal care and experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) at East Carolina University.

Spawning and fertility

After zebrafish reached their maturity at ~ 4-months of age, 10 homozygous pgrmc1/2^−/− female fish were crossed with fertility confirmed wildtype males. Number of offspring was recorded daily for a period of two weeks following a two-week acclimation period. Spawning frequency was defined as the number of times a female produce fertilized embryos in a two-week examination period.

Follicle isolation and quantification of different stage follicles

Oocyte maturation in zebrafish typically occurs prior to the onset of (day) light, while ovulation and spawning occurs within 1 hr following the onset of light. Therefore, adult females (n = 7) from different genotypes (wt and pgrmc1/2^−/−) were euthanized at 09:30am, thirty minutes after laboratory lights were turned on, by placing each fish in a lethal dose of MS-222 (300 mg/L buffered solution) for 10 minutes, then severing the spinal cord and blood supply using IACUC approved procedures. The ovaries of each fish were then immediately dissected out and rinsed in 60% L-15 media (Sigma-Aldrich, St. Louis, MO, USA) containing 15 mM HEPES (pH=7.2). Follicles of various sizes were isolated from the ovaries using fine forceps. Thereafter, ovaries were transferred to a 15-ml centrifuge tube and pipetted up and down to separate the follicles. Then the separated ovaries were transferred to a 90-mm petri dish containing 60% L-15 media. The diameter of each follicle was measured using a stereo microscope (SZX7, Olympus, Japan). The development of follicles was divided into five stages based on morphological criteria and on physiological and biochemical events (Selman et al 1993, Tyler & Sumpter 1996): Stage I (<140 μm) and II (140–340 μm) previtellogenic follicles; Stage III early vitellogenic follicles (340–690 μm); Stage IV late vitellogenic follicles (690–730 μm), which is comprised of two stages, immature Stage IVa oocytes (before germinal vesicle breakdown, i.e., GVBD) and mature Stage IVb (underwent GVBD but haven’t yet gone through ovulation); and Stage V ovulated follicles (730–750 μm), characterized as ovulated eggs with no follicular cells attached.

RNA isolation and Real-time quantitative PCR

Based on the size standard mentioned above, total RNA was isolated from Stage III (21:00), Stage IVa (6:00), and Stage IVb (08:00) follicles using the RNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) according the manufacturer’s protocol. The quantity and quality of the RNA was determined using a Nanodrop 2000 (Thermo Fisher). RNA samples were then reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Real-time quantitative PCR (qPCR) was performed using SYBR green with C1000 Touch Thermal Cycler (Bio-Rad). PCR efficiency was calculated from the equation of efficiency (EFF) = 10(−1/slope) − 1 and authentic PCR products were confirmed by analyses of melting curve, gel electrophoresis, and DNA sequencing. PCR data was analyzed using the absolute quantitation method, expressed as copies/μg RNA, and was determined using Ct values of samples and a standard curve from serial known concentrations of plasmids containing different cDNA fragment of target genes. Comparative Ct method was not used in this study because house-keeping-genes vary between different developmental stages of follicles (Liu et al 2018). The primers used in this study can be found in our previously published work (Liu et al 2018).

Western blotting

Expression of Pgr in the fully-grown Stage IVa immature follicles was confirmed by Western blot analysis using a previously developed polyclonal antibody for Pgr (Hanna & Zhu 2011b). Total protein from 10 Stage IVa follicles (06:00) that was collected directly from freshly sacrificed fish was sonicated in 100 μl of 1× SDS sample buffer (62.5 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 100 mM Dithiothreitol) on ice for about 10 short bursts (Sonic Dismembrator, Fisher Scientific). Samples were then immediately boiled for 10 minutes and stored in −20°C freezer until Western blot analysis. 10 μL of each sample was loaded onto 8% SDS PAGE gel and transferred to a nitrocellulose membrane. The membrane was first pre-incubated for 3 hrs with a blocking solution containing 5% BSA (albumin from bovine serum, Sigma A7906) in TBST (50 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.4), then with a primary antibody (Pgr, 1:250 dilution; alpha-Tubulin (Sigma, T6074), 1:3000 dilution) in the 1% BSA blocking solution overnight. The following day, the membrane was washed five times for a period of 5 minutes each with 1× TBST, incubated for 2 hr with horseradish peroxidase conjugated secondary antibody (1:5000 dilutions, goat anti-rabbit antibody for Pgr detection or goat anti-mouse antibody for α-Tubulin), and finally washed five times for a period of 5 minutes each with 1× TBST. The membranes were developed using Super Signal West Extended Dura Substrate (Pierce, Rockford, IL, USA) in a plastic wrap, then visualized using a Fluor Chem 8900 imaging station (Alpha Innotech, San Leandro, CA, USA). Protein size was determined by comparison to a biotinylated protein ladder (Cell Signaling Technology, Danvers, MA, USA) and a prestained protein ladder (Fermentas, Waltham, MA, USA). Finally, image analyzing software (ImageJ) was used to estimate relative densitometries (Schneider et al 2012).

Statistical analysis

All the results were presented as mean ± SEM. Significant differences among paired treatment groups was determined using unpaired Student’s t test and among multiple treatment group using One-way analysis of variance (ANOVA) followed by Turkey’s test (GraphPad Prism 7.0a, San Diego, CA, USA). Statistical significance was set at p<0.05.

Results

Reduced fertility in pgrmc1/2^−/− female zebrafish

To evaluate the reproductive capacities in pgrmc1/2^−/− female zebrafish, mature mutant females (n=10) at 4 months of age were mated with known fertile wildtype males during a minimum 4-week mating study period (two weeks of accommodating period followed by two weeks of quantification period). The fecundity of pgrmc1/2^−/− female zebrafish, determined as total number of embryos produced over two weeks following a two-week acclimation period, was recorded and compared to those in wildtype crossing (wildtype males crossed with wildtype females) that were treated exactly the same during the same time period. We found a significantly lower number of offspring produced by pgrmc1/2^−/− female zebrafish (pgrmc1/2^−/−, n=10, 828.5 ± 131.3, p<0.0001) in comparison to those produced by wildtype females (n=10, 1827 ± 63.2) (Fig. 1A). pgrmc1/2^−/− females also spawned with significantly less frequency (n=10, 60.71 ± 8.06%) as compared with wildtype females (n=10, 92.14 ± 1.67%) (Fig. 1B). In addition, embryos produced daily and embryos produced each time female spawned also were significantly lower in pgrmc1/2^−/− female zebrafish (Fig. 1C–1E). These results obtained in the double knockout of Pgrmc were similar as those we reported previously in the single knockout of pgrmc1^−/− or pgrmc2^−/− (Wu et al., 2018; Wu et al., 2019)

Fig 1. Reduced fertility in pgrmc1/2^−/− homozygous female zebrafish.

(A) Mutant female zebrafish produced fewer embryos over a two-week mating period than wt females. (B) Mutant females spawned with less frequency than wildtype. (C) pgrmc1/2^−/− females spawned less embryos per day. (D) pgrmc1/2^−/− females spawned less embryos each time they spawned. (E) Wildtype females produced more embryos daily than pgrmc1/2^−/− females. **, p<0.01; ****, p<0.0001.

Reduced oocyte maturation and ovulation in pgrmc1/2^−/− in vivo

To determine the possible cause for reduced fertility in pgrmc1/2^−/−, ovaries from 4-month-old zebrafish were sampled 30 minutes after lights were turned on (09:30) and the number of different staged follicles were counted. No significant differences were found in the number of early stage, immature follicles (Stage II and Stage III) in the ovaries from pgrmc1/2^−/− females in comparison to those from wildtype female fish (Fig. 2). Typically, fully grown immature follicles (Stage IVa) would have already successfully completed processes of oocyte maturation and ovulation, and no Stage IVa and Stage IVb would be found in the ovaries from wildtype females after lights were on for half an hour (Fig. 2). However, a significantly large number of these Stage IVa and Stage IVb follicles still could be observed in pgrmc1/2^−/− females (Fig. 2). These results indicate that oocytes maturation and oocyte ovulation process were affected in pgrmc1/2^−/−.

Fig 2. Quantification of different stages of oocytes in wt and pgrmc1/2^−/− female ovaries.

Significant numbers of fully-grown immature Stage IVa follicles and matured but not ovulated Stage IVb oocytes were observed in pgrmc1/2^−/− females 30 minutes after room lights were switched. At the same time point, no Stage IVa and Stage IVb follicles could be observed in wildtype females. Further, pgrmc1/2^−/− females had less Stage V oocytes.

Pgr was downregulated in pgrmc1/2^−/−

To identify the possible genes underlying the reduced ovulation found in pgrmc1/2^−/−, we examined the expression of Pgr in the Pgrmc mutants as Pgr is a key upstream regulator for ovulation. The RNA expression of pgr was significantly lower in pgrmc1^−/− single mutants in Stage III oocytes collected at 21:00, in Stage IVa oocytes sampled at 06:00 in both pgrmc1^−/− and pgrmc1/2^−/−, and in Stage IVb oocytes sampled at 08:00 in all Pgrmc mutants (Fig. 3A). The lowest RNA expression of Pgr was observed in pgrmc1/2^−/− of Stage IVa oocytes sampled at 06:00 (Fig. 3A). The protein levels of Pgr were significantly reduced in Stage IVa follicles (06:00) of pgrmc1/2^−/−, but not in pgrmc1^−/− or pgrmc2^−/− single mutant (Fig. 3B).

Fig 3. Reduced Pgr expression in the most advanced follicles of pgrmc1/2^−/−.

The night prior to spawning at 21:00, the most advanced stage of oocytes sampled at 21:00 are Stage III oocytes. These oocytes will grow further and develop into Stage IVa oocytes (fully grown but immature) at 06:00. Then, these advanced oocytes will undergo oocyte maturation to become Stage IVb oocytes around 08:00 about 1 hour prior to lights on and ovulation. (A) Expression of pgr transcripts in most advanced stage oocytes from wt or Pgrmc mutants over different developmental time points (21:00, Stage III; 06:00, Stage IVa; 08:00, Stage IVb). (B) Reduced expression of Pgr protein in Stage IVa follicles (06:00) only in pgrmc1/2^−/−. *, p<0.05; **, p<0.01; ***, p<0.001.

Reduced expression of metalloproteinase

Based on the lower expression levels of Pgr protein in pgrmc1/2^−/−, we hypothesized that metalloproteinases required for ovulation would be low in the pgrmc1/2^−/−, since Pgr regulates metalloproteinases expression during oocyte ovulation. Therefore, we collected different stages of oocytes and determined expression of six representative metalloproteinase at critical time points. Several representative metalloproteinases including adamts1, adamts8a, adamts9, and mmp2, mmp9) were significantly reduced in pgrmc1/2^−/− Stage IVb follicles (Fig. 4). Interestingly, adamts9 and mmp9 were also significantly lower in pgrmc1^−/− and pgrmc2^−/− than those in wildtype (Fig. 4).

Fig 4. Expression of metallopeptidase in Pgrmc knockouts in comparison to wt in most advanced follicles.

(21:00, Stage III; 06:00, Stage IVa; 08:00, Stage IVb.). Low adamts9 expression can be observed in Stage IVb oocytes of all genotypes (pgrmc1^−/−, pgrmc2^−/−, and pgrmc1/2^−/−). In addition, expression of adamts1, adamts8a, and mmp2 was significantly reduced in Stage IVb follicles in pgrmc1/2^−/−. Asterisks indicate a significant difference of transcripts compared to wt at the same time point. adam8b, a distintegrin and metalloproteinase domain 8b; adamts1, a disintegrin and metalloproteinase with thrombospondin type 1 motif 1; adamts8a; adamts9; mmp2, matrix metalloproteinase 2; and mmp9, matrix metalloproteinase 9. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Discussion

Pgrmc1 and Pgrmc2 have been suggested as important adaptors in mediating progesterone signaling and steroid synthesis, but their functions in vivo have not been well established. To our knowledge, this is the first evidence that Pgrmc1 and Pgrmc2 play a role in the regulation of Pgr and oocyte ovulation. We found reduced oocyte ovulation in pgrmc1/2^−/− females in vivo but not in pgrmc1^−/− or pgrmc2^−/− (Wu et al 2019, Wu et al 2018). One main cause for reduced oocyte ovulation in pgrmc1/2^−/− fish is likely due to dramatically reduced protein expression of Pgr, which is the essential regulator of oocyte ovulation. Lower protein expression of Pgr subsequently caused abnormally low metalloproteinase expression and resulted in reduced ovulation. Our results indicate an interaction between two different progestin receptor families, which operate with each other to regulate ovulation.

Low Pgr causes reduced ovulation

It is well established that Pgr is essential for ovulation across species. In agreement with other studies, pgr also increases prior to ovulation in zebrafish (Bayaa et al 2000, Hanna & Zhu 2011a, HILD-PETITO et al 1988, Liu et al 2018, Park & Mayo 1991, PRESS & GREENE 1988, Richards & Ascoli 2018). Selective PGR antagonist RU486 or CDB-2914 can reduce the number of ovulated oocytes in mice (Loutradis et al 1991, Palanisamy et al 2006). In addition, the oocytes remain trapped within follicles in Pgr^−/− mice and zebrafish because pre-ovulatory follicles are unable to undergo follicle-wall degradation (Lydon et al 1995, Zhu et al 2015). Our study demonstrated that low Pgr protein expression in pgrmc1/2^−/− zebrafish also resulted in ovulation reduction in vivo, supporting the notion that Pgr is a pivotal factor for oocyte ovulation.

Pgrmc1 and Pgrmc2 affect gene expression of Pgr

A previous study in mice showed ablation of Pgrmc1/2 did not alter the RNA expression of the Pgr (Clark et al 2016), but immunohistochemistry results showed less staining in the Pgrmc1/2^d/d uterine. Similarly, we also detected low Pgr transcription and protein level in pgrmc1/2^−/− zebrafish. The relationship between these two progesterone receptor families is still unclear. Previous research suggests a compensatory mechanism between Pgrmc1 and Pgr since higher levels of Pgrmc1 have been found in the brains of Pgr-KO female mice than in their wild-type littermates (Krebs et al 2000). Also, activation of Pgr can represses expression of Pgrmc1 during lordosis facilitation (Krebs et al 2000). In our results, we found a new relationship between Pgrmcs and Pgr; and Pgrmcs are important for normal expression of Pgr. But how Pgrmc1 and Pgrmc2 regulates the expression of Pgr remains unknown and needs further investigation. In our previous work, we showed Pgrmc1 and Pgrmc2 to be highly expressed in the follicular cells and denuded oocytes (Wu et al 2019, Wu et al 2018). In interphase cells, Pgrmc1 was distributed throughout the cell including the nucleus (Peluso et al 2014). But whether Pgrmcs can bind to transcription factors and directly affect Pgr gene expression remains unclear. PGRMC1 interacts with epidermal growth factor receptor (EGFR) (Ahmed et al 2010, Aizen & Thomas 2015, Kabe et al 2016) and membrane progesterone receptor α (mPR α) (Aizen et al 2018, Thomas et al 2014). The downstream signaling of EGFR and mPR α may result in gene expression differences of Pgr. Pgrmc1 and Pgrmc2 also co-localized to cytoplasm (Peluso et al 2014), indicating a close relationship between Pgrmc1 and Pgrmc2. Pgrmc1 and Pgrm2 may have similar, but not identical, functions because double knockout (pgrmc1/2^−/−) female zebrafish had a more severe reproductive phenotype than those found in pgrmc1^−/− or pgrmc2^−/− single knockout female zebrafish, respectively. This unknown function from Pgrmcs may explain why oocyte ovulation delay and Pgr protein decrease only happens in pgrmc1/2^−/− but not single mutants.

Lower proteinase expression causes ovulation delay

In late stage follicles, such as Stage IVa and Stage IVb, the follicular cells surrounding the oocyte synthesize a variety of factors to induce ovulation, including several kinds of metalloproteinases. This process is under the control of Pgr (Liu et al 2017). The involvement of several metalloproteinases, including members of MMP (matrix metalloproteinase), ADAM (A Disintegrin And Metalloproteinases), and ADAMTS families, has been examined (Brown et al 2010, Peluffo et al 2011, Robker et al 2000, Sriraman et al 2008). In our pgrmc1/2^−/− follicles, we observed downregulation of several metalloproteinases. Adamts1 has been identified as an important metalloproteinase exert functions in oocyte ovulation. ADAMTS1 cleaves the surrounding versican-rich matrix of oocytes and allows the release of the oocyte (Brown et al 2010, Robker et al 2000). In Adamts1^−/− mice, both ovulation and subsequent fertilization were severely impaired as a result of the versican persistence (Brown et al 2010, Mittaz et al 2004, Shozu et al 2005). Adamts1 transcripts increased in mice granulosa cells of periovulatory follicles, with this high expression being induced by hCG and progesterone. But this induction is impaired in Pgr^−/− ovaries, indicating that Adamts1 acts downstream of Pgr (Robker et al 2000). In our Pgrmc mutant lines, low adamts1 expression can only be seen in the Stage IVb oocytes of the pgrmc1/2^−/−. Besides Adamts1, Adamts9 also plays an important role in oocyte ovulation. Due to embryonic lethality in Adamts9^−/− (Dubail et al 2014, Enomoto et al 2010), the function of this protein during ovulation is unknown. Some adamts9^−/− zebrafish can survive and grow to adulthood, but the ovaries are not well developed in mutants (Carter et al 2019). Interestingly, Adamts9 expression is induced by LH and hCG in mature follicles in zebrafish, monkeys, and humans during early ovulation (Liu et al 2018, Peluffo et al 2011, Rosewell et al 2015). Adamts9 is specifically expressed in follicular cells but not in the oocytes and its expression increase significantly prior to ovulation (Liu et al 2018). In addition, the expression of adamts9 is significantly downregulated in the follicular cells of anovulatory pgr^−/− zebrafish (Liu et al 2018). The proteoglycans aggrecan and versican are known substrates of Adamts9. Therefore, Adamts9 is important for ovulation. Our results demonstrated that adamts9 expression reduced in pgrcm1^−/−, pgrmc2^−/−, and pgrmc1/2^−/− when compared to wt. It is plausible that reduced ovulation in pgrmc1/2^−/− might be due to lower level of adamts9 resulting from the lower levels of Pgr. MMPs may also play critical roles in ovulation through remodeling extracellular matrix (Cooke et al 1999). In rhesus monkies, granulosa cells exposed to LH in vitro showed elevated RNA levels of MMP9 (Duffy & Stouffer 2003). MMP9 also plays a critical role in LH-induced steroidogenesis in mouse granulosa cells during ovulation (Light & Hammes 2015). In our Pgrmc mutant lines, transcription of mmp9 showed a peak in Stage IVa oocytes and drop in Stage IVb oocytes. This mmp9 peak is prior to that of Adams and Adamts expression, indicating a different role Mmp9 play in oocyte ovulation. Compared to wt, both mmp2 and mmp9 are lower in the pgrmc1/2^−/−. In fact, the RNA expressions of adamts9 and mmp9 were also low in the pgrmc1^−/− and pgrmc2^−/−, but we did not observe ovulation delay in single-knockout Pgrmc mutants in vivo. Other proteinases such as adamts1, adamts8a, and mmp2 may also contribute to abnormal ovulation in pgrmc1/2^−/−. However, we need to interpret these gene expression results with great caution since the protein expression and enzyme activities of these proteinases are missing due to lack of antibodies and bioassays. Further studies including follicular cell specific knockout of proteinases is required to determine role of proteinases in the ovulation

Oocyte maturation delay in pgrmc1/2^−/−

In pgrmc1/2^−/−, we also found some Stage IVa oocytes after lights were on for 30 mins; and GVBD experiments also show reduced oocyte maturation in vitro. Our previous studies show the different roles that Pgrmc1 and Pgrmc2 play in oocyte maturation. Pgrmc1 promotes oocyte maturation through increased plasma localization and expression of mPRα (Thomas et al 2014, Thomas et al 2007, Wu et al 2018). Further, oocyte maturation reduced in pgrmc1^−/− in vivo and in vitro (Wu et al 2018). In contrast, the maturation rate of fully-grown immature oocytes from pgrmc2^−/− was similar to those found in wildtype zebrafish in vitro. Lower progestin synthesis enzymes were observed in pgrmc2^−/−, which may cause reduced oocyte maturation delay (Wu et al 2019). Therefore, it is likely that the oocyte maturation delay found in pgrmc1/2^−/− in vivo and in vitro is caused by both Pgrmc1 and Pgrmc2.

This is the first time it has been shown that both Pgrmc1 and Pgrmc2 are required for the regulation of Pgr signaling and function in ovulation. Losing both Pgrmcs ultimately leads to transcriptional and translational changes of Pgr. Information obtained from the present study will contribute to our understanding of the interaction between different progesterone receptors. Further research is needed to understand why Pgrmc1 and Pgrmc2 can affect the gene expression of Pgr.

Highlihts:

1. Subfertility in double knockout of pgrmc1 and pgrmc2 (pgrmc1/2^−/−) female zebrafish

2. Reduced ovulation in pgrmc1/2^−/−.

3. Reduced expression of nuclear progestin receptor (Pgr) in pgrmc1/2^−/−.

4. Reduced expression of metalloproteinases in pgrmc1/2^−/−.