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Advances in Andrology: The Basic Science Behind Fertility, October 13, 2017

11/14/2017 by Campion Fund

Over 70 scientists gathered for a meeting presented by the Campion Fund at the National Institute of Environmental Health Sciences for talks, discussion and networking on the topic of male fertility.

Plenary speaker, Haifan Lin, PhD, Yale University, presented studies of late spermatocytes and early spermatids demonstrating a novel function of gene regulation in the germline.  He and his colleagues found that piRNAs (Piwi-interacting RNAs) are involved in the epigenetic mechanism of methylation by guiding RNA to chromatin (the complex of DNA and proteins that compresses DNA into chromosomes). A bit of background promotes the understanding of this discovery.  A gene called Piwi is a fruit fly gene. Piwi stands for P-element induced wimpy testis. The human gene is called hiwi while the mouse gene is miwi. In organisms in which DNA is in chromosomes within a distinct nucleus, there are large regions that are not genes at all. These regions contain transposons or DNA sequences that will “copy and paste” to a new position within the DNA. They also include pseudogenes or DNA sequences that are similar to genes but do not code proteins, repetitive genes and non-coding genes.  Non-coding genes produce non-coding RNA (known as IncRNA) as well as PiRNas.  piRNAs are genetically conserved, consist of 26-31 nucleotides in length and are involved in gene regulation. Dr. Lin and colleagues demonstrated that degradation of spermatogenic cell specific IncRNA by PiRna is mediated by retrotransposon RNAs which also “copy and paste” and are frequently found in mRNAs targeted for degradation. Pseudogenes also regulate mRNA stability via the piRNA pathway. Along with guiding RNA to chromatin, these findings demonstrate a highly complex and global regulatory network.  In short, a novel mechanism of epigenetics.

Debra Wolgemuth, PhD, Columbia University, discussed the testis-specific BET family protein, BRDT. BET proteins are double bromodomain and extra-terminal domain proteins and are important epigenetic readers that bind to acetylated histones in chromatin and regulate transcription and modulate changes in chromatin structure.  Dr. Wolgemuth and her colleagues have demonstrated that BRDT is essential for the normal progression of spermatogenesis. Loss of BRDT function disrupts the epigenetic state of meiotic sex chromosome inactivation in spermatocytes affecting synapsis and silencing of the X and Y chromosomes. BRDT controls chromatin organization and histone modification of the chromatin attached to the synaptonemal complex

Tracy Bale, PhD, University of Maryland, discussed studies that examined the effect of paternal chronic stress on offspring in a mouse model.  In this model stressed male mice produce offspring with significantly blunted stress responses.  Males bred three months after stress exposure produced offspring with altered stress reactivity. Chronic paternal stress altered microRNA in sperm. These RNAs are a bit smaller that piRNAs and are also involved in gene regulation.  Subsequent studies suggest that the caput epididymis is involved in the reprogramming of the sperm microRNAs. Her studies present evidence that paternal experiences can have lasting changes on the germline and thus in future offspring development and demonstrates that the environment can dynamical regulate sperm epigenetic marks.

Janice Bailey, PhD, Laval University, presented work concerned with paternal exposure to chemical contaminants and their effects on future generations.  Persistent Organic Pollutants or POPs are transported north to artic regions via weather currents and contaminate the food chain. Inuit in Canada have high amounts of these chemicals in their bodies and have lifespans 13 years shorter than non-Aboriginal Canadians.  Furthermore, their risks of infant death are greatly elevated and are thought to be due to poor fetal growth, placental disorders and congenital disorders. Dr. Bailey and her team used a multigenerational rat model to show that paternal exposure to POPs disturbs the male fertility parameters and effects the development of his offspring across generations.  Sperm DNA methylation is alerted inter-and trans-generationally.  They postulate that dietary folic acid might mitigate the effects of the POPs. She presented preliminary results that indicate that folic acid interacts with POPs.

Matt Coward, MD, School of Medicine, University of North Carolina, Chapel Hill talked about the current evaluation and treatment of male factor infertility. Male factor infertility is associated with serious medical conditions, including cancer and cardiovascular mortality.  In the US 1.2 million women are seen in office visit for infertility but only 20 percent of their male partners receive a medical evaluation. The majority of male factor problems are identifiable and are reversible as advanced diagnostic testing and surgical techniques makes treatment possible for the majority of couples with a male factor contribution.  Evaluation of infertile males is essential and includes a complete detailed medical history, physical examination, semen analysis, hormone profile and in selected situations, a genetic evaluation or imaging.

Plenary speaker, Marty Matzuk, MD, PhD, Baylor College of Medicine, discussed the work in his laboratory to study genes in male germ line development and of characterizing small molecule contraceptives to target the male germ line in vivo. Over the past three decades he has developed over 100 knockout mice models.  Approximately 21% of 37 male mouse models have fertility defects.  Based on his findings in knockout mice and using CRISPR-Cas9 technology to alter both the sperm epigenome and embryo gene expression he has identified promising drug targets for contraception in men. He identified one such small molecule, JQ1 that reversibly inhibits BRDT. JQ1 is a possible male contraceptive. He utilizes DNA barcoding to screen up to 2 billion molecules in a single assay to determine other possible targets for male contraception. His research in reproduction and contraception has been funded continuously by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, a former employer of mine.

Blanche Capel, PhD, Duke University, investigated the molecular mechanisms that regulate the stable transition from male gonocytes to pro-spermatogonia. Prior to this transition, germ cells may readily form teratomas. Dnd1 or dead-end homologue is a RNA binding protein that acts as a crucial mediator of this transition.   A mutation in Dnd1 increases the incidence of testicular teratomas in mutant mice to over 90%. By comparing transcriptomes in germ cells of wild type to mutant mice Dr. Capel and her colleague found that the cells of the mutant animals continued to express pluripotency genes, SOX2, NANOG and NODAL. These cells failed to enter cell cycle arrest and did not activate critical genes of male germ line differentiation. They found that the NANOS pathway and a number of chromatin modifying enzymes were affected. This finding helps understand the role of DND1 in the transition from male gonocytes to pro-spermatogonia and have implications for both fertility and cancer development.

Christopher Geyer, PhD, East Carolina University, discussed retinoic acid responsiveness and how in regulates the fate of mammalian spermatogenesis. In neonatal male germ cells mice, postnatal day 1 prospermatogonia and postnatal day 5-6 spermatogonia differ in their responsiveness to retinoic acid. In wild type mice day 1 none of the prospermatogonia activated stra8, the retinoic acid inducible gene indicating that they have not yet been exposed to retinoic acid. By postnatal day 5-6, 20 % of the spermatogonia are STRA8+ showing that they are exposed.  When exogenous retinoic acid is added 70 %t of day 1 cells and 85 % of day 5-6 cells can respond to the retinoic acid revealing that molecular controls exist in vitro and in vivo. This suggests that molecular controls protect a subset of germ cells from retinoic acid exposure.  He described current studies to understand this mechanism.  A small subset of spermatogonia are protected from retinoic acid a represent a possible spermatogonia stem cell pool.

Jannette Dufour, PhD, Texas Tech University, presented studies exploring aspects of testis immune privilege. This immune privilege involves more than sequestering germ cells behind the Sertoli cell barrier. Sertoli cells express immunoregulatory proteins. They will survive transplantation by inducing regulatory regulatory T cells and M2 macrophages. In addition, Sertoli cells survival is attributed to a significant decrease in proinflammatory cytokines and an increase in anti-inflammatory cytokines, immunomodulatory factors such as Il-10, CDC4+ and CD25+ and Foxp3+ T cells.   Sertoli cells survive and protect co-transplanted allografts and xenografts.  She demonstrated that when Sertoli cells were engineered to produce insulin they significantly decreased blood glucose levels after transplantation into diabetic mouse models. The cells survived and produced insulin for more than 70 days after transplantation. These studies strengthen the possible use of Sertoli cells in cell-based therapy.

Tony De Falco, PhD, Cincinnati Children’s Hospital Medical Center, discussed his work on understanding the differentiation of fetal Leydig cells.  Leydig cells are steroidogenic and produce testosterone. They arise from the progenitor cells in the interstitial compartment of the testis.  His work demonstrates that vascular-mesenchymal interactions are critical morphogenetic forces in testes cord formation. Dr. Falco and colleagues have shown that few Leydig cell progenitors are maintained in the absence of blood vessels. Perivascular interstitial cells undergo Notch signaling suggesting that there is vascular niche for fetal Leydig cell progenitors.  In studying the critical interactions between vasculature and interstitial progenitors, their studies begin to provide insights into male infertility and other reproductive conditions resulting from Leydig cell defects.

Poster abstracts presented by twelve scientists provided an interesting and lively poster session.  The studies presented represented science conducted by individuals in a number of countries including the United States, (NIEHS, Virginia Commonwealth University, Baylor College of Medicine, Duke University, University of Illinois Urbana, University of Massachusetts) China (Huazhong University, Wuhan University) and France (Université de Nice-Sophia).  Topics presented included: Estrogen-containing contraception association with increased 25-hydroxy vitamin D; Critical regulation of GLIS3 in early postnatal mouse spermatogenesis; The essentialness of IFT27 (intraflagellar transporter protein 27) in spermatogenesis; Loss of COUP-II2 (NR2F2) affects fetal testicular development;  FOXL2 reprogramming of somatic cell fate in adult and fetal testis; The opposing functions of DAZL and ILF2 in protein 1 and 2 mRNA in mouse spermatogenesis; Ovarian cell aberrant expression of Sf1 disrupts ovarian somatic cell function and fertility; Dnd1 mutation transcription alterations in germ cells perturb key components in male germline establishment; Reduced IFT172 expression in male germ cells reduced sperm number, motility and male fertility in mice;  SIX1 oncoprotein is necessary for abnormal uterine basal cells development in DES exposure in neonatal mice;  Single mutation using CRISPR/cas9 system in meiosis-expressed Gene I results in impaired spermatogenesis and infertility in mice; Novel gene and pathway identification in sexual reproductive tract differentiation in mice.

 

NIEHS News provides a summary of the meeting along with photographs of the participant. To see this summary, click on this link: https://factor.niehs.nih.gov/2017/11/science-highlights/reproductive-science/index.htm