Decision letter: The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Follicle rupture, the final step in ovulation, utilizes conserved molecular mechanisms including matrix metalloproteinases (Mmps), steroid signaling, and adrenergic signaling. It is still unknown how follicles become competent for follicle rupture/ovulation. Here, we identify a zinc-finger transcription factor Hindsight (Hnt) as the first transcription factor regulating follicle's competency for ovulation in Drosophila. Hnt is not expressed in immature stage-13 follicle cells but is upregulated in mature stage-14 follicle cells, which is essential for follicle rupture/ovulation. Hnt upregulates Mmp2 expression in posterior follicle cells (essential for the breakdown of the follicle wall) and Oamb expression in all follicle cells (the receptor for receiving adrenergic signaling and inducing Mmp2 activation). Hnt's role in regulating Mmp2 and Oamb can be replaced by its human homolog Ras-responsive element-binding protein 1 (RREB-1). Our data suggest that Hnt/RREB-1 plays conserved role in regulating follicle maturation and competency for ovulation. https://doi.org/10.7554/eLife.29887.001 eLife digest The release of an egg from the ovary of a female animal is a process known as ovulation. Animals as different as humans and fruit flies ovulate in largely similar ways. Yet the systems involved in controlling ovulation are still not well understood. An egg cell develops within a collection of cells that help the egg to form properly. Together, this unit is called a follicle. During ovulation, connections between the egg and the rest of the follicle break down and the egg is eventually ejected. Ovulation happens in response to a hormone signal from the brain. In humans, this hormone is called luteinizing hormone, whereas in flies it is called octopamine. Specialized protein molecules on the surface of the follicle cells receive these hormone signals, but can only cause ovulation in mature follicles. It was not clear what allows only mature follicles to ovulate. Deady et al. have now used the fruit fly Drosophila melanogaster to examine ovulation to identify how the process is controlled. The results showed that a protein called Hindsight primes follicle cells for ovulation. When a follicle reaches its final stage (called stage 14 in flies), the gene for Hindsight becomes active and produces the protein. This protein then activates other genes. One of the activated genes makes a protein that receives the hormone signal, while another makes a protein that breaks down follicle cells and allows the egg to be released. The findings of Deady et al. reveal that Hindsight is needed for ovulation in flies. Further experiments then showed that the gene for equivalent human protein can be transplanted into flies and can still prime follicles for ovulation. This indicates that the genes in humans and flies may perform the same tasks. Studying ovulation is an important part of understanding female fertility and could help scientists to understand more about human reproduction. These results may also lead to new contraceptives and improved approaches for treating infertility. https://doi.org/10.7554/eLife.29887.002 Introduction Ovulation is a complex process of releasing fertilizable oocytes from mature follicles and is essential for animal reproduction (Espey and Richards, 2006). To ensure successful ovulation, a follicle must be developed to full maturity to be competent to receive an ovulatory stimulus and to activate proteolytic systems for follicle rupture. Several proteolytic systems have been found to regulate follicle rupture in vertebrates, including matrix metalloproteinase (Mmp), plasminogen activator/plasmin, and ADAMS-TS (Curry and Smith, 2006; Takahashi et al., 2013). In addition, a surge of luteinizing hormone (LH) serves as a master regulator to initiate the ovulation event and activates the EGF/EGFR-Ras-MAPK signaling pathway to propagate the ovulatory signal from outer granulosa cells to inner cumulus cells in the preovulatory follicles (Conti et al., 2012; Fan et al., 2009, 2011, 2012; Hsieh et al., 2007). However, molecular mechanisms coupling the Ras-MAPK pathway to the activation of proteolytic systems for follicle rupture are largely unknown. Ovulation in Drosophila utilizes conserved molecular mechanisms and involves a follicle rupture process to release mature oocytes from the ovary. Drosophila have two ovaries, connected at their posterior ends by bilateral oviducts (Figure 1). Each ovary contains ~16 ovarioles, where egg chambers are assembled in the germarium at the anterior and develop through 14 characteristic stages toward the posterior end (Spradling, 1993). Each egg chamber contains one oocyte and 15 nurse cells surrounded by a layer of somatic follicle cells. In stage-14 egg chambers (also named mature follicles), all nurse cells are degraded, leaving an oocyte surrounded by follicle cells; Matrix metalloproteinase 2 (Mmp2) is upregulated in posterior follicle cells (Figure 1; Deady et al., 2015). In addition, Oamb (octopamine receptor in mushroom body), encoding an α-adrenergic receptor-like G-protein-coupled receptor for octopamine (OA), is also upregulated in all follicle cells of stage-14 egg chambers (Lee et al., 2003; Deady and Sun, 2015). OA, released from terminal nerves that innervate ovaries, activates Oamb receptor in stage-14 follicle cells, which induces calcium rise and activates Mmp2 (Deady and Sun, 2015; Heifetz et al., 2014; Middleton et al., 2006; Monastirioti, 2003). Mmp2 enzymatic activity leads to degradation of posterior follicle cells and release of the encapsulated oocyte (called follicle rupture; Figure 1; Deady et al., 2015). The rest of the follicle cells remain at the end of the ovariole to form a corpus luteum (Deady et al., 2015). Local adrenergic signaling has also been suggested to regulate mammalian ovulation but no molecular mechanisms have been illustrated (Kannisto et al., 1985; Schmidt et al., 1985). In parallel to progesterone signaling in mammalian ovulation, ecdysteroid signaling is also activated in stage-14 follicle cells and is essential for Drosophila ovulation; ecdysteroid signaling modulates OA/Oamb-induced Mmp2 activation, but does not affect Oamb expression nor Mmp2 expression (Knapp and Sun, 2017). Thus, it is currently unknown what induces Mmp2 and Oamb expression in stage-14 follicle cells and how these follicles become fully competent for ovulation. Figure 1 Download asset Open asset An illustration of Drosophila ovulation process. The female reproductive system, consisting of two ovaries, oviduct, uterus, seminal receptacle, and a pair of spermathecae and parovaria, was depicted in the cartoon. Two representative ovarioles with different staged egg chambers were highlighted in the right ovary. Oocytes and nurse cells are in yellow. Mmp2 expression is shown in green and Mmp2 activity is shown in red. https://doi.org/10.7554/eLife.29887.003 The zinc-finger transcription factor Hindsight (Hnt; encoded by gene pebbled) contains 14 C2H2 zinc-finger domains and is homologous to mammalian Ras-responsive element-binding protein 1 (RREB-1). Both Hnt and RREB-1 bind to similar DNA sequences, and human RREB-1 can functionally replace Hnt in attenuating expression of nervy and hnt itself in Drosophila salivary gland (Ming et al., 2013). RREB-1 functions downstream of the Ras-MAPK pathway to either suppress or promote Ras target genes in multiple tissues including colon, thyroid, and pancreatic cancers (Kent et al., 2010, 2013; Mukhopadhyay et al., 2007; Thiagalingam et al., 1996; Zhang et al., 2003). Hnt is expressed in a variety of tissues in development and plays multiple developmental roles including control of embryonic germ band retraction (Yip et al., 1997; Reed et al., 2001), regulation of retinal cell fate and morphogenesis (Pickup et al., 2002; Wilk et al., 2004; Pickup et al., 2009; Oliva and Sierralta, 2010; Oliva et al., 2015), maintenance of tracheal epithelial integrity (Wilk et al., 2000, 2004), and differentiation of spermathecae and intestinal stem cells (Sun and Spradling, 2013; Baechler et al., 2015). Hnt is also expressed in follicle cells of stage 7-10A egg chambers, where it functions as a downstream target of Notch signaling to suppress Hedgehog signaling and to induce the mitotic/endocycle transition (Sun and Deng, 2007). Hnt continues its expression in anterior follicle cells throughout late oogenesis. In contrast, Hnt expression in mainbody follicle cells is downregulated from stage 10B to stage 13 and re-upregulated in stage-14 (Deady et al., 2015), where its role is unknown. Moreover, few downstream targets of Hnt have been identified and its relationship to Ras signaling is also unknown. Here, we characterized the dynamic expression of Hnt in stage-14 follicle cells. By using molecular and genetic tools, we demonstrated that Hnt expression in stage-14 follicle cells is essential for follicle rupture partly by upregulation of Oamb and Mmp2 expression in these follicle cells. Thus, Hnt functions as an essential transcription factor to prime follicles to be competent for follicle rupture/ovulation. In addition, Hnt's role in follicle rupture can be replaced by human RREB-1. Our data, along with the involvement of Ras-MAPK signaling in mammalian ovulation, lead us to propose that Hnt/RREB-1 has a conserved role in regulating follicle rupture/ovulation downstream of Ras-MAPK signaling pathway. Results Dynamic expression of Hindsight in stage-14 follicle cells Hnt is not expressed in stage-13 follicle cells except those at the anterior region; however, it is upregulated in all stage-14 follicle cells and the corpus luteum (Deady et al., 2015). Upon closer examination, we found three distinct patterns of Hnt expression throughout stage-14 egg chambers: (I) high Hnt expression in anterior and posterior but low/no Hnt in the middle follicle cells ('A/P-Hnt' egg chambers; Figure 2A and F, Figure 2—figure supplement 1A–B); (II) high Hnt expression in all follicle cells ('high-Hnt' egg chambers; Figure 2B and G); (III) low Hnt expression in all follicle cells ('low-Hnt' egg chambers; Figure 2C and H; also see Figure 2—figure supplement 1C–D). To determine the developmental sequence of the aforementioned three types of stage-14 egg chambers, we analyzed the expression patterns of Hnt against the expression of a stage-14 follicle-cell Gal4 driver (44E10-Gal4; renamed as FC1 for simplicity) (Deady and Sun, 2015) and the number of nurse cell nuclei in these egg chambers. 86% of A/P-Hnt egg chambers had medium-level GFP expression driven by FC1, while more than 73% of high-Hnt and 80% of low-Hnt egg chambers had high-level GFP expression (Figure 2A–D). This indicates that A/P-Hnt egg chambers are the youngest, which is consistent with the observation that A/P-Hnt egg chambers typically have more residual nurse-cell nuclei than the other two types of egg chambers (Figure 2E). In addition, high-Hnt egg chambers still contained one or two nurse-cell nuclei, while low-Hnt egg chambers typically did not contain nurse-cell nuclei and were skinner and dehydrated (Figure 2E and Figure 2—figure supplement 1D). Because nurse-cell nuclei are progressively degraded starting around stage-12 by a non-cell-autonomous mechanism to generate fully matured egg chambers, which have no nurse cell nuclei and are dehydrated (Drummond-Barbosa and Spradling, 2004; Timmons et al., 2016), the above analysis demonstrates that high-Hnt egg chambers are at the intermediate stage, while low-Hnt egg chambers are the most mature egg chambers. Figure 2 with 1 supplement see all Download asset Open asset Hindsight expression in stage-14 egg chambers. (A–C) Hnt expression (red in A-C and white in A'-C') in A/P-Hnt (A), high-Hnt (B), and low-Hnt (C) egg chambers. FC1 expression (FC1 Gal4 driving UAS-eGFP, FC1 > GFP) is shown in green (A–C) and white in (A"–C"). Nuclei are shown by DAPI in blue (A–C). (D–E) Quantification of FC1 expression (D) and residual nurse cell nuclei (E) in A/P-Hnt, high-Hnt, and low-Hnt egg chambers. The number of stage-14 egg chambers analyzed is noted above each bar. (F–H) Hnt expression (red in F-H and white in F'-H') in A/P-Hnt (F), high-Hnt (G), and low-Hnt (H) egg chambers. FC2 expression (FC2 > GFP) is shown in green (F–H) and white (F"–H"). Nuclei are shown in blue (F–H). (I–J) Quantification of FC2 expression (I) and residual nurse cell nuclei (J) in A/P-Hnt, high-Hnt, and low-Hnt egg chambers. (K) A schematic cartoon shows the temporal pattern of Hnt, FC1 and FC2 expression in stage-14 egg chambers. FC1-related graphs are colored green and FC2-relaed graphs are colored blue. https://doi.org/10.7554/eLife.29887.004 This conclusion was further supported by additional analysis using a late stage-14 follicle-cell Gal4 driver (47A04-Gal4; renamed as FC2 for simplicity) (Deady and Sun, 2015). Consistent with the previous result, both A/P- and high-Hnt egg chambers had no or minimal GFP expression driven by FC2, while low-Hnt egg chambers had highest GFP expression and fewest nurse-cell nuclei (Figure 2F–J). Altogether, these analyses demonstrate that Hnt is first upregulated in posterior follicle cells, filled in across the entire egg chamber, and then overall downregulated in follicle cells of fully matured egg chambers (Figure 2K). Therefore, we propose to categorize stage-14 egg chambers into three distinct stages and rename A/P-, high-, and low-Hnt egg chambers as stage-14A, stage-14B, and stage-14C egg chambers, respectively (Figure 2K). Hindsight in stage-14 follicle cells is required for normal ovulation The dynamic Hnt expression in stage-14 follicle cells prompted us to investigate its function in follicle maturation and ovulation. To bypass the early requirement of Hnt in follicle cell differentiation (Sun and Deng, 2007), we used RNA interference (RNAi) to deplete Hnt expression specifically in stage-14 follicle cells with FC1 or FC2 Gal4 driving UAS-hntRNAi expression. While FC1 started to be expressed in stage-14A follicle cells, it was weak to deplete Hnt expression in stage-14A follicle cells (Figure 3—figure supplement 1A–D), but became progressively more efficient in older follicle cells with two independent hntRNAi lines (Figure 3A–D and Figure 3—figure supplement 1E–H); more than 80% of stage-14C egg chambers had no detectable Hnt expression in their follicle cells. In contrast, FC2 started to be expressed in stage-14B follicle cells and most effectively depleted Hnt expression in stage-14C follicle cells except with hntRNAi2, which only showed strong reduction in ~43% of egg chambers (Figure 3E–H and Figure 3—figure supplement 1I–P). Figure 3 with 2 supplements see all Download asset Open asset Hindsight expression in stage-14 follicle cells is essential for normal ovulation. (A–D) Hnt expression (green) in control (A) and hntRNAi (B–C) stage-14C egg chambers with FC1. FC1 expression (FC1 > RFP) is shown in red. Inserts are high magnification of Hnt expression in squared areas. The quantification of Hnt expression (categorized as high-Hnt, low-Hnt, A/P-Hnt, and None-Hnt) in stage-14C egg chambers is shown in (D). The number of stage-14C egg chambers (selected according to no nurse-cell nuclei/high FC1 expression) analyzed is noted above each bar. (E–H) Hnt expression (green) in control (E) and hntRNAi (F–G) stage-14C egg chambers with FC2. FC2 expression (FC2 > RFP) is shown in red. Inserts are high magnification of Hnt expression in squared areas. The quantification of Hnt expression in stage-14C egg chambers is shown in (H). The stage-14C egg chambers are selected according to no nurse-cell nuclei/high FC2 expression. (I–J) The quantification of egg laying (I) and mature egg chambers in each female's ovaries after egg laying (J) in control or hntRNAi females with FC1 (green bars) or FC2 Gal4 (blue bars). The number of females is noted above each bar. (K–L) The egg-laying time in control or hntRNAi females with FC1 (K) or FC2 (L). Also see Supplementary file 1. *p<0.05, **p<0.01, ***p<0.001. https://doi.org/10.7554/eLife.29887.006 Females with RNAi-mediated hnt depletion in stage-14 follicle cells (named hntRNAi females for simplicity) were then assayed for egg-laying ability. hntRNAi females laid significantly fewer eggs than control females after mating (Figure 3I). This phenotype was manifested by using both stage-14 follicle-cell Gal4 drivers and with two independent hntRNAi lines, which demonstrates that Hnt expression in stage-14 follicle cells was essential for normal egg laying. The decrease in egg-laying number was not caused by an oogenesis defect, as plenty of stage-14 egg chambers were observed before and after egg-laying experiments (Figure 3J and Figure 3—figure supplement 2). The egg-laying process consists of ovulation (the release of egg from the ovary into the oviduct), egg transportation through the oviduct, and oviposition (the release of egg in the uterus to the outside environment). To determine which step in the egg-laying process was affected in hntRNAi females, we utilized our previously developed method to estimate the average time required for each step in the egg-laying process (Sun and Spradling, 2013; Deady and Sun, 2015; Knapp and Sun, 2017). Consistent with our previous data, control females spent 12–14 min to ovulate an egg, less than a minute to transport egg through the oviduct, and eight - 10 min to hold an egg in the uterus and oviposit (Figure 3K–L and Supplementary file 1). In contrast, hntRNAi females spent more than 25 min to ovulate an egg, which was significantly longer than the control females (Figure 3K–L and Supplementary file 1). These data demonstrate that Hnt in stage-14 follicle cells is required for normal ovulation. Hindsight in stage-14 follicle cells is necessary for OA-induced follicle rupture Ovulation consists of a breakdown of posterior follicle cells and a subsequent rupture of oocyte into the lateral oviduct (Figure 1), which is induced by octopaminergic signaling and can be recapitulated in an ex vivo culture system (Deady and Sun, 2015). The requirement of Hnt for normal ovulation led us to hypothesize that Hnt is required for OA-induced follicle rupture. Consistent with this idea, about 45% stage-14 egg chambers isolated according to FC1 expression from control females ruptured in response to OA stimulation, whereas fewer than 10% of egg chambers from hntRNAi females ruptured in response to OA stimulation (Figure 4A–D). In addition, more than 85% stage-14 egg chambers isolated according to FC2 expression from control females ruptured in response to OA stimulation (Figure 4E and H), consistent with the fact that FC2 is expressed in more mature egg chambers than FC1 (Figure 2). In contrast, egg chambers isolated from hntRNAi1 or hntRNAi2 females with FC2 ruptured at the rate of 10% and 33%, respectively (Figure 4F–H). Consistent with this result, follicles isolated from hnt transheterozygous mutant females also showed significant reduction in OA-induced follicle rupture in comparison to control follicles (Figure 4—figure supplement 1). These results demonstrate that Hnt is required in stage-14 follicle cells for follicle rupture. Figure 4 with 1 supplement see all Download asset Open asset Hindsight is required for OA-induced follicle rupture. (A–D) Representative images show control (A) and hntRNAi (B–C) egg chambers with FC1 after three-hour culture with 20 μM OA. Quantification of OA-induced follicle rupture is shown in (D). (E–H) Representative images show control (E) and hntRNAi (F–G) egg chambers with FC2 after three-hour culture with 20 μM OA. Quantification is shown in (H). Egg chambers were isolated according to FC1 > RFP (red in A-C) or FC2 > RFP (red in E-G) expression. Bright-field images of the egg chambers are shown in blue, and ruptured egg chambers are marked by white arrowheads. The number of egg chambers is listed above each bar. ***p<0.001. https://doi.org/10.7554/eLife.29887.009 Hindsight is essential for Mmp2 activity in posterior follicle cells of stage-14 egg chambers Mmp2 is essential for follicle rupture, and its enzymatic activity is activated by OA stimulation (Deady and Sun, 2015). To determine whether Hnt regulates follicle rupture by influencing Mmp2 activity, we assayed Mmp2 enzymatic activity in egg chambers from control and hntRNAi females after OA stimulation ex vivo. After a three-hour incubation with OA, ~60% of control egg chambers isolated according to FC1 expression had posterior gelatinase activity (Figure 5A and D), whereas only ~25% of hntRNAi egg chambers had posterior gelatinase activity (Figure 5B–D). In addition, about 90% of control egg chambers isolated according to FC2 had posterior gelatinase activity, in contrast to 25% and 47% of hntRNAi1 and hntRNAi2 egg chambers, respectively (Figure 5E–H). The proportion of follicles with posterior gelatinase activity was correlated to the proportion of follicles that ruptured, and both were significantly decreased in hntRNAi egg chambers, which strongly supports that Hnt controls follicle rupture by regulating Mmp2 activity. Figure 5 Download asset Open asset Hindsight regulates Mmp2 activity in stage-14 follicle cells. (A–C) Representative images show OA-induced Mmp2 activity (green) in control (A) and hntRNAi (B–C) egg chambers with FC1 after three-hour culture with OA. Egg chambers with posterior Mmp2 activity are marked by arrowheads. Quantification is shown in (D). (E–H) Representative images show OA-induced Mmp2 activity (green) in control (E) and hntRNAi (F–G) egg chambers with FC2 after three-hour culture with OA. The quantification is shown in (H). (I) A diagram shows the three categories of basement-membrane (BM) configurations (according to Vkg::GFP expression) of follicle cells in isolated stage-14 egg chambers. When a line connecting the posterior-most Vkg edges bisects the oocyte, it is defined as an open-BM configuration, whereas when the line does not bisect the oocyte, it is defined as a broken-BM configuration. The intact-BM configuration is defined as intact, continuous Vkg::GFP throughout the posterior of the egg chamber. (J–J') A control egg chamber identified according to FC2 >RFP expression shows the open-BM configuration. (K–K') An egg chamber with overexpression of Timp shows the intact-BM configuration. (L–L') An Mmp2RNAi egg chamber shows the broken-BM configuration. (M–N) hntRNAi egg chambers show intact-BM (M–M') and broken-BM (N–N') configurations. (O) Quantification of BM configurations of FC2-expressing egg chambers with respective genotypes. The number of egg chambers analyzed is noted above each bar. ***p<0.001. https://doi.org/10.7554/eLife.29887.011 To further support this notion and to avoid the possibility that the above observed phenomenon is an artifact of ex vivo culture, we determined whether Hnt indeed regulates Mmp2 activity in vivo. One of the known substrates of Mmp2 is the basement-membrane (BM) protein collagen IV, encoded by viking (vkg), during imaginal disc morphogenesis and fatbody dissociation at pupal development et al., 2007; et al., Vkg is in the of follicle cells throughout oogenesis and we it could be a of Mmp2 as found that of FC2-expressing egg chambers had Vkg protein in a posterior had Vkg protein in a posterior and had Vkg protein at their posterior end (Figure and When of matrix metalloproteinase encoding an of enzymatic was in stage-14 follicle cells using FC2, the BM configuration was toward intact-BM configuration (Figure and that activity is for the degradation of Vkg at the posterior end of stage-14 egg chambers. In addition, RNAi-mediated Mmp2 depletion in stage-14 follicle cells showed a similar as overexpression of less effectively (Figure and that Mmp2 at for the Vkg hnt depletion in stage-14 follicle cells also BM configuration toward and intact-BM configuration as Mmp2 depletion (Figure Altogether, these data demonstrate that Hnt regulates Mmp2 activity, which is for Vkg degradation at the posterior end of stage-14 egg chambers during ovulation. Hindsight is required for Mmp2 expression in posterior follicle cells at stage 14 OA to Oamb receptor in stage-14 follicle cells, which leads to a rise of and subsequent activation of Mmp2 (Deady and Sun, 2015). To the mechanism of Hnt in regulating Mmp2 activity, we to determine whether Hnt with pathway downstream of In comparison to OA, was to induce more than control egg chambers to rupture at the end of a three-hour culture, whether egg chambers were isolated according to FC1 or FC2 expression (Figure and In contrast, was still not to induce follicle rupture in hntRNAi egg chambers those with FC2 driving hntRNAi2 Figure and that it was to induce rise in follicle cells (Figure supplement The of hntRNAi egg chambers to stimulation indicates that Hnt regulates downstream of rise in the the normal response to but response to OA in hntRNAi2 egg chambers with FC2 indicates that Hnt also regulates of rise in the pathway. Consistent with this idea, OA was not to induce rise in hntRNAi egg chambers with FC2 (Figure supplement Hnt is first upregulated in posterior follicle cells (Figure where Mmp2 is expressed (Deady et al., 2015), and then across the entire follicle cells (Figure where Oamb is expressed (Lee et al., 2003; Deady and Sun, 2015), we hypothesize that Hnt regulates both Mmp2 and Oamb expression in stage-14 follicle cells. Figure with 2 supplements see all Download asset Open asset Hindsight regulates Mmp2 expression in stage-14 follicle cells. of egg chambers isolated according to FC1 (A–D) or FC2 (E–H) to rupture in three and Representative images show control and and hntRNAi and egg chambers after the images of the egg chambers are shown in blue, and white ruptured egg chambers. Quantification of rupture response to OA or to is shown in and Representative images show expression (green in and white in in control or hntRNAi egg chambers with FC2 Gal4 Nuclei are with DAPI and shown in blue to posterior follicle cells, and oocytes are in Quantification of expression in control and hntRNAi egg chambers using FC1 or FC2 Quantification of Mmp2 in hntRNAi egg chambers with FC1 or FC2 (O) *p<0.05, **p<0.01, ***p<0.001. 1 Download asset This be in does support may still the for Download as Download as Download as of driven by FC2 in follicles with stimulation (FC2 > 2 Download asset This be in does support may still the for Download as Download as Download as of driven by FC2 in hntRNAi1 follicles with stimulation (FC2 > 3 Download asset This be in does support may still the for Download as Download as Download as of driven by FC2 in hntRNAi2 follicles with stimulation (FC2 > 4 Download asset This be in does support may still the for Download as Download as Download as of driven by FC2 in follicles with octopamine stimulation (FC2 > 5 Download asset