REGULATION OF THE ACROSOME REACTION BY THE

TRANSMEMBRANE ADENYLYL CYCLASE

SEPTEMBER 2012

DOUGLAS HADDAD, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST

M.Sc., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Pablo Visconti

Capacitation prepares mammalian sperm to undergo a process known as the acrosome reaction, which enables them to penetrate the zona pellucida. The standard method of measuring the acrosome reaction has been over the years the staining of the acrosome and visual counting using light or fluorescence microscopy. In this study we explored the intracellular signaling that results in the acrosome reaction using a novel method. This method employs the use of transgenic mice that contain green fluorescent protein, GFP, in the sperm acrosome. The quantity of sperm either containing or lacking GFP is precisely and rapidly quantified with flowcytometry. Currently little is known about the signaling processes that lead to the acrosome reaction. It has been proposed that this reaction is regulated by the cAMP activated guanine nucleotide exchange factor, Epac. In human sperm, stimulation of this pathway leads to an increase in acrosomal exocytosis. Furthermore, previous studies from our laboratory indicated that Gα_s is present in the mouse sperm anterior head. These results suggest that in mouse sperm, the cAMP pathway leading to the acrosome reaction could be stimulated by Gα_s associated with a transmembrane adenylyl cyclase. In this study we first validated the ability of known reaction inducers to increase the rate of the acrosome exocytosis in mouse sperm. Progesterone, solubilized zona pellucida and calcium ionophore A23187 all showed to be very effective at inducing the acrosome reaction in mouse sperm. We then investigated the role of the cAMP pathway using a battery of cAMP agonists and antagonists. We observed that stimulation of the cAMP dependent pathway through the transmembrane adenylyl cyclases, using forskolin, inhibit the increase in acrosome reaction induced by soluble zona pellucida, progesterone and the calcium ionophore A23187. This inhibitory effect was observed only when forskolin was used before the start of capacitation. Consistent with this observation, addition of cAMP analogues including an Epac specific cAMP analogue 8-pCPT-2’-O-Me-cAMP can also inhibit the increase in acrosome reaction by progesterone. This inhibition was seen only when the pathway was stimulated from the beginning of capacitation. Altogether, these data suggest that transmembrane cyclases are involved in the regulation of mouse sperm acrosome reaction.

The first half of this thesis investigates the role of Smyd3 as a possible maternal-effect gene, which was identified through an mRNA-based expression assay. Using a Smyd3 conditional allele to remove the function of the protein, we were able to confirm successful allelic recombinations but were unable to confirm the absence of the protein. Due to a lack of a phenotype, two conclusions were made: 1) If we did disrupt Smyd3 function, the lack of a phenotype indicates that Smyd3 is not a maternal-effect gene, and is not required for embryonic viability or adult fertility. 2) It is possible that newly-annotated transcripts are responsible for the necessity of Smyd3, and that the conditional allele used did not disrupt the function of Smyd3.

The second half of this thesis uses a reverse genetic RNAi screen that identified Wdr74 as being required for the critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos, blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse.

To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture.

The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis.

The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.

As an alternative we have turned to magnetic separation of human mammary epithelial cells from breast milk. Initial studies with these cells, which are detailed in chapter one, show that a breast milk sample provides sufficient epithelial cells to isolate high quality RNA for gene expression analyses or genomic DNA for methylation analysis of multiple genes. Using quantitative RT-PCR of RNA collected from these samples we detected differences in the mRNA levels for six genes known to be down regulated in breast cancers: BRCA1, p16, CDH1, TMS-1, GSTPi, and SFRP1. Additionally using methyl-specific PCR (MSP) we assayed for a small panel of genes frequently methylated in cancer and found them to be unmethylated in the few breast milk samples examined. However, given the small number of CpG sites which can be assayed by the MSP technique it is not surprising that methylation was not detected in disease-free subjects.

With methods for collecting breast milk samples and processing them for genetic material established we turned to a more comprehensive study of DNA methylation in larger population of donors which is detailed in chapter two. Utilizing a highly sensitive and highly quantitative methylation analysis technique known as Pyrosequencing we examined age-related methylation patterns for RASSF1A, TMS-1, CDH1, SFRP1, GSTPi, and CRBP1 in genomic DNA purified from exfoliated epithelial cells magnetically enriched from breast milk (n=111) and whether the protective effects conveyed by early pregnancy could be partly due to decreases in DNA hypermethylation. Although firm answers about early pregnancy were inconclusive based on our sample pool, this body of work lays down a solid foundation for future studies.