The Role of Bec-1 in Oocyte Quality

In women, oocytes arrest development during prophase of meiosis I and remain inactive for years. Over time the quality and quantity of these oocytes decreases; therefore, fertility declines as human females age. C. elegans display a similar trend showing a decrease in the quality of oocytes as they grow older. Oocyte quality is assayed in C. elegans by determining the fraction of oocytes that produce viable eggs following fertilization. A decreased ability to eliminate genetically defective oocytes could account for this drop in oocyte quality. However, mutations that only prevent cell death in response to DNA damage have little effect on embryonic lethality. By contrast, mutations in C. elegans that block all cell deaths or germline specific cell deaths result in a drastic early decline in oocyte quality. Furthermore, mutants in which cell death could still function but the recycling of the corpse was prevented, lowered the quality of oocytes. This evidence indicates that developmental programmed cell deaths allow the proper allocation of resources to growing germ cells. This function of apoptotic cell death is the main contributor to oocyte quality rather than the removal of cells with damaged DNA. Therefore, oocytes compete for resources in the germline and apoptosis is vital for the efficient distribution of these resources required for proper oocyte development. This experiment sought to develop a method to study the influence a specific gene involved in the cell death pathway ultimately has on oocyte quality. bec-1 is linked to apoptotic cell death, since inactivation of bec-1 triggers ced-3/caspasedependent programmed cell death. There is an interaction between bec-1 and ced-9 that is required for ced-9 to function properly. Without bec-1 excessive cell death occurs. bec-1 is also a gene involved in the intracellular process of autophagy. Autophagy maintains the balance between synthesis, degradation and recycling of cellular products. As seen in the case of apoptosis, this reallocation of resources may be required for proper oocyte development and quality. These findings make bec-1 an extreme gene of interest for its role in oocyte quality. Since bec-1 mutants are not viable, to study its effect on oocyte quality its activity must be reduced only in the germline. To perform germline specific RNAi, the rrf-1 strain was used. rrf-1(pk1417) mutants lack an RNA dependent RNA polymerase necessary for an RNAi to be successful in the somatic cells but not in the germline. It was found that bec-1 activity can be reduced specifically in the germline of rrf-1(pk1417); bec-1(RNAi); fog-2 (oz40) animals. This result allows the direct study of the role bec-1 plays in oocyte quality in C. elegans. INTRODUCTION As women age, the quality and quantity of their oocytes decline. This results in an increased chance of having a child with birth defects or experiencing a miscarriage. This also makes it much more difficult for an older woman to become pregnant [1]. The National Survey of Family Growth found that the probability of a woman achieving pregnancy in one year or less was significantly higher in women who were less than thirty years of age compared to women older than thirty-five. A similar study found that the monthly probability of conception leading to a live birth remained optimal until the age of thirty-one. The monthly probability of conception after thirty-one progressively decreased thereafter. Once a woman has reached the age of thirtyM. JUNG: THE ROLE OF BEC-1 IN OOCYTE QUALITY -2eight, probability of conception has dropped to one quarter of that in women less than thirty years old [2]. The oocytes are of lower quality in aging women resulting in eggs that are less likely to mature and eventually be fertilized. Additionally, among older women, there is a larger fraction of oocytes that have chromosomal abnormalities [2]. These abnormalities can be attributed to defects in recombination and cohesion during meiosis [3]. C. elegans can be used to study how oocyte quality is regulated during aging [4]. Oocyte quality can be assayed by determining the fraction of oocytes that produce viable eggs following fertilization. This characteristic of C. elegans is the most crucial and pertinent for the scope of this experiment. Furthermore, the time required for C. elegans fully to develop is minimal. XX animals are self-fertile hermaphrodites, making crosses very easy. XO animals are considered male. These males can be used in crosses where self-fertilization of the XX animal is not desired. Additionally, C. elegans are transparent, making for easy analysis of their body form under a microscope. At 15oC, the first 60-80 germ cells in the hermaphrodite develop into spermatocytes. This results in approximately 300 sperm. Germ cells following the initial production of spermatocytes develop into oocytes. The female gonad consists of two symmetrical U-shaped tubes connected to a uterus at the center of the animal. As germ cells develop, they move from the distal tip towards the proximal portion of the gonad and meiosis ensues. The germ cells increase in size as they mature, but more than half of the developing oocytes undergo apoptosis. The reason for these germ cell deaths is debated. However, proper regulation of these cell deaths is imperative for the normal development of healthy ooctyes [5]. In other words, when cell death is improperly regulated, oocyte quality declines. Fourteen genes have been identified that function in the cell death pathway (Figure 1). This pathway may be broken down into four main steps. Step number one is the decision of the cell to undergo programmed cell death. Step two is characterized by the actual killing of the cell. Step three is the engulfment of that cell and step four its degradation [6]. The same genetic pathway is used to carry out all cell deaths in C. elegans. However, hermaphroditic development in C. elegans has very different developmental origins. For this reason, there may be several ways to induce cell death that is cell-type-specific. Ultimately, different signals that induce cell death may converge to activate the same pathway [7]. The genes ced-3 and ced-4 are required for all cell deaths to occur. They are the key genes in step two where the actual killing of the cell happens. Without these genes, no cell death may take place [7]. ced-9 protects C. elegans from undergoing programmed cell death [6]. A gain of function mutation in ced-9 results in the survival of cells that would die normally. Inactivation of ced-9 results in the death of many cells that would normally survive. There is an interaction between the bec-1 gene and ced-9 that is required for ced-9 to function properly. Without bec-1 excessive cell death occurs [6]. Engulfment of the dying cell takes place next. Mutations in the seven genes (ced-1,2,4,6,7,8,10) block this engulfment process. The gene nuc-1 is required to degrade the DNA of the dead cell. In mutants that lack nuc-1 activity, the cells die and are engulfed, but the DNA of the dead cells is not degraded (Figure 1) [6]. TCNJ JOURNAL OF STUDENT SCHOLARSHIP VOLUME XII APRIL, 2010 -3Oocyte quality declines in C. elegans as they age as in human women [8],[5]. A decreased ability to eliminate genetically defective oocytes could account for this drop in oocyte quality. If this were the case, then specifically preventing all cell death of oocytes with damaged chromosomes would result in a high rate of embryonic lethality. These cell deaths require CEP-1 which acts through egl-1 to regulate ced-9 activity. Any loss-of-function mutation in these genes prevents cell death in response to DNA damage but does not affect physiological germ cell deaths. However, only 12% of eggs in these mutant mothers die before hatching. Physiological germ cell deaths require ced-3 and ced-4 but are not affected by mutations in egl-1 or ced-9(gf) [5]. A more likely scenario is that these numerous cell deaths seen in the germ line are required to provide nutrients to other oocytes so that they may develop properly. Mutations in C. elegans that block all cell deaths or germline specific cell deaths result in a drastic early decline in oocyte quality. Mutations that only prevent somatic cell deaths have no effect on oocyte quality. Furthermore, mutants in which cell death could still function but the recycling of the corpse was prevented lowered the quality of oocytes [5]. Therefore, developmental programmed cell deaths in the germline permit the proper allocation of resources to growing germ cells which can explain the drop in oocyte quality. This function of apoptotic cell death is the main contributor to oocyte quality rather than the removal of cells with damaged DNA. The evidence shows, as C. elegans age, the competition for resources increases and apoptosis is vital for the efficient distribution of these resources required for proper oocyte development [5]. The study of specific genes involved in the cell death pathway could further elucidate the precise factors and mechanisms that are required for proper oocyte development. bec-1 is a gene that is directly involved in the cell death pathway [9]. bec-1 is a highly conserved gene from C. elegans to mammals making it a gene of great interest in the study of infertility. It has been linked to apoptotic cell death in C. elegans, when bec-1 binds the antiapoptotic protein ced-9. As previously mentioned, the improper regulation of cell death causes a drop in oocyte quality. However, bec-1 also plays a crucial role in the intracellular process of autophagy. Autophagy is the degradation of the cell’s own components in which bec-1 is required for the formation of the autophagosome [10]. Since autophagy influences both apoptosis and the reallocation of resources in the cell, it might help regulate oocyte development and quality. M. JUNG: THE ROLE OF BEC-1 IN OOCYTE QUALITY -4The two roles bec-1 plays in autophagy and apoptotic cell death make it a gene worth investigating for its possible role in oocyte quality. However, in mutants where bec-1 was deleted, approximately 90% of the animals underwent embryonic arrest [11]. Therefore, to study the role of bec-1 in oocyte quality, an animal is required whose gene activity is reduced only in the germ line. This can be accomplished by performing a modified RNAi experiment. In this study, double stranded RNA for bec-1 was injected into the distal portion of the gonad of rrf1(pk1417); fog-2(oz40) mutants. fog-2 is a sex determination gene. XX C. elegans are normally hermaphrodites; therefore, they produce both sperm and eggs. A balance between the levels of fem-3 and tra-2 determines if spermatogenesis or oogenesis takes place. A high ratio of fem-3/tra2 promotes spermatogenesis. Meanwhile, a low ratio of fem-3/tra-2 promotes oogenesis. In XO animals, her-1 inhibits tra-2 so that sperm is produced continuously (Figure 2). In XX animals, her-1 activity is absent. tra-2 is negatively regulated post-transcriptionally by gld-1 and fog-2 to permit the onset of hermaphrodite spermatogenesis. Therefore, fog-2 mutants lack fog-2 activity necessary to inhibit tra-2 post-transcriptionally. With this uninhibited tra-2 activity, fem-3 levels are low and oogenesis persists (Figure 2). This makes these XX animals essentially female [12]. This permits control of the exact time the eggs of these animals are fertilized. The rrf-1 gene codes for an RNA dependent RNA polymerase. RNA dependent RNA polymerases copy messenger RNAs and spread the amplified sequences throughout the cell. This amplification and spread of the injected dsRNA is required for a successful RNA interference experiment [13]. The double stranded bec-1 mRNA that is injected into the gonad of the worm requires the rrf-1 gene in the somatic cells for its amplification and dispersal. However, rrf-1 is one member of a family of RNA dependent RNA polymerases. Other RNA dependent RNA polymerases are present in the germline allowing rrf-1 mutants to be bec-1 deficient in the germline only. Therefore, rrf-1 mutants lack the RNA-dependent RNA polymerase necessary for an RNAi to be successful in the somatic cells, but not in the germline [14]. Thus, these animals TCNJ JOURNAL OF STUDENT SCHOLARSHIP VOLUME XII APRIL, 2010 -5should be deficient in bec-1 in the germline, but normal in other tissues. If so, this technique would allow direct study of the effect bec-1 has on oocyte quality. MATERIALS AND METHODS C. elegans were handled using standard methods [15]. The animals were maintained on NG plates at 15oC (NG Agar: 6 g NaCl, 9 g KH2PO4, 1.5 g K2HPO4, 12 g tryptone, 60 g Agar, and 1 ml cholesterol in ethanol (15 mg/ml) were added to 3 L dH2O and autoclaved). Female animals were aged to the early adult stage before being used for microinjection. A PCR was run to amplify a DNA template for bec-1. Primers were designed for bec-1 that have T7 promoter sequences. The forward primer sequence was 5’TAA TAC GAC TCA CTA TAG GGA GAG TTT GTA ATG ATT GCT CTG ACG CT-3’. The reverse primer sequence was 5’TAA TAC GAC TCA CTA TAG GGA GAT CCA TGT CGA TGC CAT TAC GAC GA -3’. The DNA was purified using a QIAquick column. The concentration of the template was measured using a spectrophotometer. The DNA concentration obtained was 75 nanograms/microliter. To confirm that the correct fragment was obtained, an agarose gel (0.8%) was run. The predicted size of the DNA strand was 644 nucleotides. The gel showed a band of approximately that length, when compared to a 100 base pair ladder (Figure 3). Figure 3. Agarose gel (.8%) showing the fragment size of the bec-1 DNA (sample 2) compared to a 100 base pair ladder. The DNA strand measured 644 nucleotides. The DNA concentration obtained was 75 nanograms/microliter. A Megascript reaction (Ambion), was then conducted to transcribe the DNA into its corresponding RNA using this DNA template. The reaction was allowed to run overnight. The RNA was purified using a Megaclear kit. The single-stranded RNA was then annealed by being heated to 70 oC and returning to room temperature in approximately twenty-five minutes. The concentration, which was once again measured using a spectrophotometer, was approximately 800 nanograms/microliter. The microinjection technique was used to inject the double-stranded RNA into the distal portion of the gonad of young adult female C. elegans. First, fog-2(oz40) mutants were injected. They were then transferred to new plates to lay their eggs. The progeny were screened for bec1(RNAi) phenotypes, were many of which observed. Some of these defects were seen in the ventral nerve cord, the intestine, the developing vulva, the pharynx, and the gonad. However, the most distinct and easily observed was the pharynx defect. The pharynx in these mutants did not develop properly. Defective worms were photographed at 1000v DIC optics. Forty of these M. JUNG: THE ROLE OF BEC-1 IN OOCYTE QUALITY -6animals were scored. Next, the bec-1 double-stranded RNA was injected into rrf-1(pk1417);fog2(oz40) mutants. Once again, the injected worms were transferred to new plates to await egg production. Forty of the progeny of the injected rrf-1(pk1417);fog-2(oz40) worms were screened for defects. Additionally, forty fog-2(oz40) animals that were not injected were screened for defects as a control.