Fourth Annual DNA Grantees' Workshop
Wednesday, June 25, 2003
MORNING SESSION
Sperm Cell Selection System for Forensic DNA Analysis of the Male Component: Proteomics and Genomics of Sperm-Specific Markers
John C. Herr
Biography
MR. STAPLES: Our next speaker is Dr. John Herr. He is professor of cell biology and director of the Center for Research in Contraceptive and Reproductive Health at the University of Virginia School of Medicine. He has published over 150 scientific papers and is an active inventor, having filed 36 patent applications. Dr. Herr is a former president of the American Society of Reproductive Immunology. Please welcome Dr. Herr.
DR. HERR: Coming to this meeting, I just want to thank NIJ for inviting me, because there's a lot of energy here and I think forensic science is really coming into its own right now. I think we can all help advocate for such activity by contacting our congressman and trying to push new bills ahead. It's a very exciting time, and I'm pleased to be part of this. Herr: Slide 1
You've already heard a lot about genes, but I'm going to talk about proteins. In particular, I'm going to focus on proteins that can be used to help identify sperm. The aim of our project was to design a sperm-binding magnetic bead or a magnetic filament. The idea was to couple these particles with monoclonal antibodies to sperm proteins and then evaluate the efficiency of this method for separating sperm DNA from the DNA of all the contaminating cells that you find in sexual assault evidence. In particular, we had targeted a monoclonal antibody called S19. We were growing it in bulk, and this was going to be our target antibody-antigen system for isolating sperm. Herr: Slide 2
The monoclonal antibody S19 is against a sperm surface antigen called sperm agglutination antigen 1 (SAGA–1). We chose this antigen because it's located on all surface domains: the head, the midpiece, and the tail. This antigen is acquired by the sperm in the epididymis as the sperm transits and is stored in the epididymis before ejaculation. The S19 monoclonal is an IgG1 isotype. Herr: Slide 3
This is just a phase image of human sperm. Herr: Slide 4
This is a fluorescent image where the monoclonal antibody S19, after having reacted with human sperm, stained the sperm surface. When you extract sperm proteins and stain them on this two-dimensional gel, most of the sperm proteins have isoelectric points over here [indicating] in the 4 to 10 range. You don't see the 10 range here because it's cut off at about 6, but most of the sperm proteins are over here [indicating] are in this isoelectric point range. Herr: Slide 5
The SAGA–1 antigen seen in this western blot is a set of very acidic proteins, from about 15 to 25 kDa and an isoelectric point of about 2.5 to 3. So this is what the SAGA–1 protein actually looks like. It's a GPI-anchored protein and has a number of different carbohydrate groups on it that contribute to this polymorphism.
We grew the S19 IgG in gas-permeable bags, seen here, and we ran the IgG over a protein G column where we isolated a lot of this monoclonal antibody. This is what you see when we do the purifications. Notice the purified heavy chain and purified light chain from the purification. Herr: Slide 6
We went ahead and coupled these with magnetic particles. In this case, we were using 120- to 160-nanogram paramagnetic particles. We were in fact able to get the particles to bind to the sperm surface, as you can see. First in this DIC phase contrast image, this is what the particles looked like when they bound to the sperm surface. Because we had a fluorescent antibody involved, we could actually come back in and show exactly where the antibody was on the particle. Herr: Slide 7
So, we were able to create these beads with the antibody and show that they could bind to the sperm surface. But when it came to actually pulling out evidence and looking at sperm extracted from swabs, we made a rather astounding discovery, because I think this was the first time that sperm recovered from dried swabs went through an ultrastructural analysis. We basically discovered that the majority of sperm had lost their plasma membranes and their outer acrosomal membrane. Herr: Slide 8
To prove this, we studied different populations of sperm by electron microscopy. In condition one, we looked at fresh semen that was fixed and embedded for electron microscopy. In the second condition, we took fresh semen and air-dried it onto cotton swabs at room temperature, stored it for 6 days, rehydrated it, eluted it, and then processed it for electron microscopy. For condition three (this study group) we collected postcoital samples on cotton swabs, stored them for several months, and then rehydrated, eluted, and processed them for electron microscopy. Herr: Slide 9
This shows you the result of those three groups. This is what a normal sperm looks like. The nucleus is intact, and there's a nice intact acrosome with both outer and inner acrosomal membranes surrounding the sperm head. On the fresh sample, you can see that the sperm membranes have exfoliated and that much of the acrosome is gone. And if you look at the postcoital sperm swab, virtually no membrane is left adherent to the sperm head. Herr: Slide 10
We did some quantitation—what's called a morphometric analysis—of the intact sperm plasma membrane on the postcoital swabs. We just measured the percent of intact membrane, basically the length of the intact sperm membrane over the length of the perimeter of the sperm head. Herr: Slide 11
This is a graphical representation of what happens to sperm when you pull them off of cotton swabs. Fresh sperm have almost 100 percent of their plasma membranes intact. Sperm from swabs have lost about 90 percent of their plasma membranes, and postcoital swabs have lost about 95 percent. Herr: Slide 12
Obviously, this has implications for isolating sperm using various cell surface markers: Herr: Slide 13
- The sperm plasma membrane is disrupted with the current methods for collection, storage, and recovery of sexual assault evidence.
- Molecules in the sperm plasma membrane may not be the best targets for forensic immunoselection.
- Other sperm antigens that are not located on the sperm plasma membrane require discovery and characterization as candidate targets.
- Current methods of collecting sexual assault evidence may need to be changed in order to recover membrane-intact sperm.
Since we talked about this last year, a number of people have contacted us and have begun projects to develop better strategies, including vaginal lavage. For those of you who are attempting to new methods of sexual assault evidence collection in an attempt to preserve sperm plasma membranes, please e-mail us at klk4P2@virginia.edu, so we can give you S19 monoclonal antibodies to work with. Herr: Slide 14
The rest of the talk is going to center on one the major new targets of non-plasma-membrane antigens that we discovered. Herr: Slide 15
To appreciate what we're doing, I just want to review the membrane and the compartments of the sperm head. This is in a human spermatid sitting in a Sertoli cell, which is seen here just as it begins to mature in the testis. This is the nucleus and the chromatin is beginning to condense. A nuclear envelope surrounds the nucleus. A subacrosomal domain can be seen here in white. Then there is an inner acrosomal membrane, an acrosomal matrix, an outer acrosomal membrane, a small domain between the plasma membrane and the acrosome, and the plasma membrane itself. So those are the membrane compartments that we deal with in thinking about the sperm head.
Because the plasma membranes and the outer acrosomal membranes are frequently disrupted on samples, we thought that the molecules exposed in the inner acrosomal membrane, the perinuclear cisternae, and the nuclear envelope might prove useful targets for immunoselection. Herr: Slide 16
There are two points to consider as you think about the theoretical characteristics that you would be after in attempting to achieve an immunoselection. Obviously, the target antigen must be exposed and accessible to antibody-mediated binding and be retained on sperm recovered from postcoital swabs. Also, to limit cross-reactivity with other cell types, you have to identify a target protein that shows a sperm-specific pattern of gene expression. We started searching for molecules that would meet these two criteria. Herr: Slide 17
With this, our project goals changed: Herr: Slide 18
- Identify novel sperm-specific molecules.
- Characterize their subcellular localization.
- Develop new monoclonal antibody immunoreagents to the new targets.
- Determine if the markers are in fact retained on sperm from the postcoital swabs.
Our approach combined proteomics, genomics, bioinformatics, cell biology, and developmental biology. Herr: Slide 19
The idea was to find a testis-specific pattern of gene expression, something that appeared postmeiotically. The protein I'm going to now show you is called Sperm Protein Associated with the Nucleus on the X chromosome (SPAN–X), and this protein is only present in spermatids and sperm. It's associated with the sperm nuclear envelope. We have antibodies to it now, and this marker is retained postcoitally on the sperm that are recovered from swabs. I'll show you the proof of each of these points. Herr: Slide 20
This is the human X chromosome and this is a probe for the SPAN–X genes. These are the two chromatids and you can see the sister chromatid staining here at the locus X27.1. Herr: Slide 21
If you go into the human genome and look at the contigs that are in this X27.1 region, you will find that there are six SPAN–X genes. Three of them go in one orientation and the other three are oriented in another orientation. They are called SPAN–XA, SPAN–XB, SPAN–XC, SPAN–XD, and SPAN–X. The SPAN–XB family are actually duplicates of the SPAN–XA family. So although there are six separate genes in the human genome for this family, there are in fact four separate proteins, four unique proteins that are made. Herr: Slide 22
The proteins are listed here. SPAN–XA is a 97 amino acid protein. Its mass is about 11 kDa and its pl (molecular mass) is about 5. SPAN–XB, –XC, and –XD are 103 amino acid proteins. SPAN–XB, which is the longer form, basically differs by this insertion, which is sitting right here. Herr: Slide 23
All of these sequences have a tripartite nuclear localization signal, which is seen boxed in these areas. It's these nuclear localization signals that target it to the nucleus during the production of the protein.
Now, this is just a cartoon to remind you a little bit about how sperm are made in the testis. There are spermatogonia that are the stem cells. They differentiate into spermatocytes, which undergo meiosis, and form spermatids, which differentiate from a round spermatid into this marvelous elongated cell that makes its way into the outer world with a condensed nucleus and a flagellum. Herr: Slide 24
This is a cross section of a human testis. It shows the basement membrane and the spermatogonia. When you have an antibody, which we made to the SPAN–X proteins, you can see that the cells that stain are located at the luminal side of the seminiferous epithelium and are in fact the round spermatids that stain. This indicated that the protein is a postmeiotic marker. It appears only in the round spermatids after the meiotic events have occurred. Herr: Slide 25
Here is another cartoon to remind you of the transformation that happens when spermatids mature into mature sperm. The nucleus is very open in a spermatid. The acrosome begins to form in a little vesicle. It's a little circular structure by the Golgi apparatus. Herr: Slide 26
During the sperm maturation process, the nucleus gets a lot darker and begins to elongate. This occurs as the histones are replaced with protamines and the whole genome is condensed. As the acrosomal structure flattens and elongates, it covers two-thirds of the anterior sperm head. Meanwhile, the flagella is differentiating and the midpiece mitochondria are developing.
So these are some of the processes that are going on during what we call spermiogenesis—the actual transformation of a round spermatid into a mature sperm.
Now, the reason I just showed you that is these are pictures of SPAN–X's localization in human spermatids during the differentiation of a spermatid into a mature sperm. The staining of SPAN–X is in red and the staining of another marker that we've created, a marker for the acrosome, is in yellow. Herr: Slide 27
So this is a round spermatid with a little acrosomal vesicle. The acrosomal vesicle is stained with a monoclonal antibody called MSH5, which is a specific protein for acrosomal marking, and the nuclear envelope is stained with the SPAN–X antibody, which is a specific marker for the analysis envelope of sperm and spermatids.
As the different stages of the differentiation of the spermatids proceed, you can follow both the flattening and development of the acrosome with this SP10 monoclonal antibody marker and the differentiation of the nuclear envelope with the SPAN–X marker. You also can see that the SPAN–X marker remains segregated to the part of the nuclear envelope that is not involved in the area below the acrosome, that is, the non-acrosomal regions of the nuclear envelope. Now, after elongation of the sperm head occurs, which you see here and in this step [indicating], the SPAN–X marker begins to segregate at the base of the head of the human sperm.
Now, we studied the expression of this gene in a whole host of human organs. This just shows you the extraction by a northern blot of RNA from peripheral blood lymphocytes, the colon, the small intestine, ovary, testis, prostate, and thymus. You can see the message for SPAN–X is a 0.6-kilobase message specifically in the testis. All these other organs were studied similarly by dot blot methods and only testis showed expression. Therefore, SPAN–X is indeed a testis-specific marker. Herr: Slide 28
This finding was reconfirmed by an RT–PCR (reverse transcriptase polymerase chain reaction) analysis of mRNAs from a whole host of tissues. This is the SPAN–X amplimer and you can see it's only present in the lane where the human testis is and it's absent in all the other organs in the body. Herr: Slide 29
So, at the protein level, when you run a Silverstein of all the proteins in the sperm over this PI range from 4 to 5, the SPAN–X family is found down here. In this little boxed area, as you can see, there are four genes being expressed and they show a slight set of isoforms, probably due to some slight modifications. You can get up to about 15 to 20 separate charge variants in these very high resolution gels. Herr: Slide 30
What are the SPAN–X phenotypes as you stain human sperm? This just shows you the range of how the SPAN–X protein appears. Sperm have a small crater that sometimes appears within the nucleus. Here, you can see SPAN–X within that crater, and there's the fluorescence and the superposition of the two of them. Here's a localization of it at the base of the head, and here's a SPAN–X staining area right at the head base. Then here's another example where the crater is much larger, and you can see the SPAN–X localization right over the nucleus. In sperm that have retained quite a bit of their cytoplasmic droplet, virtually the entire cytoplasmic droplet stains positive for the SPAN–X protein. Herr: Slide 31
So sperm are obviously a heterogeneous population in an ejaculate, and the SPAN–X protein similarly shows the different patterns depending on how much cytoplasmic droplet is there, how the nucleus formed, and the like.
This just shows you by ultrastructure some of the regions where the SPAN–X protein is located. Here, at the base of the head, is a whorl-like structure called the redundant nuclear envelope. After formation and condensation of the nucleus, the sperm rolls up a lot of its nuclear envelope into this redundant nuclear envelope. It's thought that after fertilization, this redundant nuclear envelope serves as a source of membrane when the nucleus decondenses prior to fertilization. The gold particles indicate the location of the SPAN–X protein in the base of the sperm head. Herr: Slides 32 and 33
Similarly, if you look at the nuclear craters, these areas that don't condense within the sperm nucleus, there is a lot of SPAN–X protein sitting in those craters, and that's what's responsible for the fluorescent dot that you see in the nucleus.
We stained postcoital samples with the antibodies and the SPAN–X, and this antigen is recoverable. Here's a region where you have a number of sperm. These are vaginal epithelial cells, and you can see the SPAN–X staining patterns of the sperm that are recovered from swabs. So, it does not undergo degradation and is retained on postcoital sperm. Herr: Slide 34
We've made a set of new monoclonal antibodies to this SPAN–X family. This is a western blot where we've run out a human sperm. Here's a series of new antibodies—D5, A1, A9, G6, B4—that are staining the SPAN–X family and are being compared to the original antibody that we produced to the recombinant protein. Herr: Slide 35
Here we've run out expressed recombinant SPAN–X on a gel, and each of those monoclonal antibodies recognizes the recombinant protein. We now have, then, a set of new monoclonal immunoreagents to this marker. Herr: Slide 36
To summarize, we present to you a new gene family, a sperm protein associated with the nucleus, encoded by the X chromosome and consisting of six loci and four unique genes, SPAN–XA through –XD, which are present in the human genome at Xq27.1. These SPAN–X proteins are expressed postmeiotically in the testis, and at least 17 little isoforms have been identified. Herr: Slide 37
You might note that this is one of the few postmeiotic genes involved in spermiogenesis that's not associated here with the Y chromosome, but rather the X chromosome is expressing a protein specifically involved in the spermeogenic process.
The SPAN–X mRNAs are in fact testis-specific, and in round spermatids, the proteins localize to the nucleus, to the acrosomal free domains of the nuclear envelope, and as the acrosome biogenesis progresses in condensing spermatids. SPAN–X localizes to the acrosomal free domains.
In ejaculated sperm, we see a number of phenotypes involving the nuclear craters and the redundant nuclear envelope. This SPAN–X protein persists on the sperm that are recovered from postcoital swabs, and with this panel of monoclonal antibodies, we have a new marker for fluorescence use, so we're now going to try and conjugate these to beads and go back and use a magnetic separation.
I'd like to thank some of the people in the Virginia group: Ken Klotz, who is here, and you can talk to him and me if you're interested in this project, and Margaretta Alietta. We had several independent summer student interns, Maria Chamorra, working on this. There's a postdoctoral fellow in the group, Pam Shoppe, who has helped to make the monoclonals, and I also want to thank Jeff Ban and Susan Greenspoon at the Division of Forensic Science in Richmond. Thank you all very much. Herr: Slide 38