Methods and Reagents

1. Computer-assisted quantification of fetal oocytes in whole-mount ovaries

Have you ever attempted to count oocytes in mouse fetal ovaries? Like ALL of them. Over and over again to get it right. Safia Malki did exactly that for several years while studying fetal oocyte attrition. In the end, she did not like it that experience. Sectioning is not always perfect. Some sections are lost or not informative. The method requires significant hands-on time.

So Safia has come up with an idea to apply a then-recently developed scale method of tissue clearing to quantification of immunostained fetal oocytes. She was later joined by a graduate student Marla Tharp who helped her to optimize the protocol and write the paper.

Since the publication of the paper, Safia and Marla continued to use and optimize the method. Recently, they have observed that the source and presumably purity of Urea is of critical importance for the Scale method performance. They are investigating the issue in further details and will be posting information on good and bad urea batches from various manufacturers.

 

2. FACS-assisted enrichment of spermatocytes from all substages of meiotic prophase.

Spermatogenesis is a complex process. Meiosis is a specialized cell cycle of germ cells that generates haploid gametes. It is the defining feature of germ cells and takes a considerable time to complete. Following the germ cell’s commitment to meiosis, it spends quite some time in meiotic prophase to exchange genetic information between homologous parental chromosomes and to prepare for two rounds of chromosome segregation. While meiosis has been studied for a long time in numerous organisms, the better understanding of the dynamics of gene expression and epigenetic changes in meiotic prophase in mammals such as mice is still necessary to improve our knowledge of meiosis in humans. Despite the abundance of male germ cells, it is not trivial to accomplish fine separation of germ cells from different steps (or substages) of meiotic prophase. Over the last few years, former Master’s student Ina Soh and graduate student Valeriya Gaysinskaya set out to optimize the method for the isolation of enriched cell populations. Their efforts led to two publications written by Valeriya that provide detailed description of the procedure in wild type and mutant mice. If you have any questions with the method (we know it can be tricky), please contact Valeriya (gaysinskaya at ciwemb.edu) for advice.

 

3. Anti-mouse LINE-1 ORF1p rabbit polyclonal antibodies

This fantastic reagent was developed and characterized by Sandy Martin in 1991. As the piRNA field took off in 2006, this polyclonal antibody has become very popular following our first description of Maelstrom knockout male mice.   In addition to reproducing patterns of ORF1p expression in wild type male germ cells described by Martin group in 1994, we observed exceptionally strong ORF1p signal in Maelstrom mutant spermatocytes (meiotic prophase I male germ cells).

In recent years, we have obtained most of the remaining serum from Sandy Martin and affinity purified ORF1p antibodies (thanks to Pavol Genzor). Over the past few years we have shared this reagent with over 60 laboratories all around the world.

If you are interested in using this reagent, feel free to contact us. We will provide you with an aliquot sufficient for several immunofluorescence and Western blot experiments. We can also provide an aliquot of testicular protein lysate from Maelstrom null mice that you can use as a positive control for Western blots.

However, please note that:

a) This antibody does not crossreact with human L1 ORF1p.

b) Please confirm L1 expression by RT-PCR first. Given the limited amounts of this reagent at the moment, it should not be used to test your ideas regarding L1 expression. It will be used best to refine expression pattern and/or cell type specificity in complex tissues.  We discourage the use of this reagent for cultured cells - RT-PCR is more than sufficient and more quantitative to determine if L1 is expressed in your favorite model.

c) Over the years, preparing antibody shipments to Australia and Japan has become prohibitively time consuming. We are talking dozens of emails, documents and notarized affidavits going back and forth between us and various parties over several weeks (!). One way to address this issue will be if one of the requesting laboratories would agree to import large number of tubes with antibody to their respective country and serve as a local distributor of this wonderful reagent to their countrymen.

Exciting news regarding Abcam anti-LINE-1 ORF1p monoclonal rabbit antibody EPR21844-108 (ab216324)

Abcam recently developed Anti-mouse LINE-1 ORF1p monoclonal rabbit antibody.  Marla Tharp and Chiara De Luca tested it in IF and WB, respectively, and we can confidently say that this is a superb reagent! We would go as far as to state that it exceeds the polyclonal rabbit L1 ORF1p antibodies that we purified from the rabbit sera (kindly provided by Sandy Martin) and have been distributing to the field over the past decade. Critically, the availability of this reagent from Abcam is great news to our colleagues throughout the world as it will bypass the need for the massive amount of paperwork on their and our sides.

Here are our results using this antibody with Maelstrom mutant testicular sections and protein lysates: