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Brinster Laboratory of Reproductive Physiology

 Our research has involved studies on mammalian germ cells and early embryos. Initially, Ralph Brinster on transgenesiswe developed a culture system and manipulation techniques for mouse eggs that are the foundation for subsequent mammalian egg and embryo experiments in the field, including nuclear transfer and in vitro fertilization of human eggs.

We then used these methods to show that mouse blastocysts can be colonized by foreign stem cells and result in chimeric adults, which led to the development of embryonic stem cells. Subsequently, we used these culture and manipulation techniques to develop transgenic mice. In recent years, our research has focused on male germline stem cells, and these studies demonstrated that spermatogonial stem cells (SSCs) from a fertile male mouse can be transplanted to the testes of an infertile male where they will colonize the seminiferous tubules and generate donor cell-derived spermatozoa, thereby restoring fertility.

In addition, SSCs of mice and other rodents can be cultured in vitro and their number increased, and the SSCs can be frozen and preserved for long periods. The ability to culture, transplant and cryopreserve SSCs makes the germline of individual males immortal. The transplantation and freezing methods are readily transferrable to the SSCs of all mammalian species.

Ralph Brinster in ScienceHowever, a culture system for SSCs of nonrodent species has proven to be difficult to develop, and published reports of success have not been independently confirmed and are not universally accepted. Therefore, in recent studies we have attempted to develop a reliable system to culture human SSCs, which is essential to preserve and expand for later use the SSCs of prepubertal boys who will receive germ cell destroying treatment for cancer.

As part of these studies, we are establishing the genes and regulating mechanism used by mouse and human SSCs to survive and replicate, which will contribute to the understanding necessary for human SSC culture and expansion. In the long term, a culture system will also allow the development of techniques to support SSC differentiation in vitro with production of spermatozoa capable of fertilizing eggs.

In addition, the SSC assay system provides a powerful technique in which to test the conversion of somatic cells to functional SSCs. Over the past 10 years, we and others have identified transcription factors and micro RNAs that play key roles in SSC self-renewal. In current research, we plan to use this information to reprogram somatic cells into germ cells, specifically SSCs. The transplantation assay provides an unequivocal conformation of this reprogramming for a single cell.

Moreover, it allows for the identification of gene activation during the differentiation process in vivo and production of progeny from sperm produced from reprogrammed cells. In the future, the approach could be used to address fertility problems in humans and possibly the correction of genetic defects.

This research is supported by grants from National Institutes of Child Health and Human Development and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.

Mary AvarbockMary Avarbock, Brinster Laboratory

  • Research Specialist & Laboratory Manager
  • Email: avarbock@vet.upenn.edu 


Male germline stem cell preservation.
Before treatment for cancer by chemotherapy or irradiation, a boy could undergo a testicular biopsy to recover stem cells. The stem cells could be cryopreserved or, after development of the necessary techniques, could be cultured. After treatment, the stem cells would be transplanted to the patient's testes for the production of spermatozoa. The critical roadblock to widespread clinical use of this approach is the absence of culture techniques. 

Brinster: Male Germline Stem Cells



Clarence FreemanClarence Freeman, Brinster Lab
Animal Research Technician

Rosie Naroznowski, Brinster Lab 
Rose Naroznowski
Animal Research Technician

 



Testis spermatogonial stem cell transplantation method. A single-cell suspension is produced from a fertile donor testis (A). The cells can be cultured (B) or microinjected into the lumen of seminiferous tubules of an infertile recipient mouse (C). Only a spermatogonial stem cell can generate a colony of spermatogenesis in the recipient testis. When testis cells carry a reporter transgene that allows the cells to be stained blue, colonies of donor cell–derived spermatogenesis are identified easily in recipient testes as blue stretches of tubule (D). Mating the recipient male to a wild-type female (E) produces progeny (F), which carry donor genes. Genetic modification can be introduced while the stem cells are in culture.

Brinster Lab: Germline Stem Cells 


 
Nilam SinhaNilam Sinha, Brinster Lab
Research Associate & Post-doctoral Fellow
Email: nilams@vet.upenn.edu

 

Studies on genes controlling spermatogenesis. Histological sections of a mouse testis showing germ cell differentiation and gradual appearance of spermatids and spermatozoa following birth (dpp is days postpartum) and an adult testis. In the histological sections (A,B,C), the red color represents the gradual appearance of protamine, which is present in spermatids and spermatozoa as they are produced following birth. The red stain is from an antibody for green florescent protein (GFP), which is expressed at the same time as protamine, because it is a transgene regulated by the protamine promoter and used as a marker for protamine in these experiments. Therefore, there is no red stain at 15 dpp (A), a small amount at 26 dpp (B), and in all spermatids and spermatozoa at 60 dpp (C), which exactly mimics the appearance of protamine in germ cells. In the adult testis (D), the green color is from GFP fluorescence under control of the protamine promoter. The variation in the intensity of the green in the adult testis represents the variation in the content of protamine at different stages of spermatogenesis in the adult testis. These mice, in which only spermatids and spermatozoa express GFP, are used in our experiments to study germ cell differentiation and determine the genetic regulation of spermatogenesis. 

Nilam Sinha, Gene studies. Brinster Lab


 
Harris HuangHarris Huang, PhD, Brinster Lab
Research Associate & Post-doctoral Fellow
Email: hahuang@vet.upenn.edu

Reprogramming somatic cells to germ cells. Somatic cells, such as mouse embryonic fibroblast (A) and hair follicle stem cell (B) are being reprogrammed into spermatogonial stem cells through lenti-viral introduction of selected transcription factors. After successful reprogramming, converted cells should look morphologically similar to spermatogonial stem cells (SSC) in culture (C). The original cells taken from the mouse will have a fluorescence marker (GFP for example) linked to a promoter for a SSC specific gene.  The cells will fluoresce upon successful conversion and when exposed to a specific wave length of light. Function of converted cells will be evaluated through transplantation (D).  If colonization is extensive, the testis will have many fluorescent tubules, and the male can be mated to a female to produce fluorescent pups that contain the genes of the mouse from which somatic cells were grown (E). If the amount of spermatogenesis is small, for example a single colony, spermatozoa can be harvested and used to fertilize an oocyte by intra-cytoplasmic sperm injection. Gene modification or correction could be performed before conversion of the somatic cell to an SSC. 

Harris Huang: somatic to germ cells 

 


 
Kathryn VolarichKathryn Volarich, Brinster Lab
Administrative Assistant & Office Manager to
R. L. Brinster, V.M.D., Ph.D.
Richard King Mellon Professor
  of Reproductive Physiology
Department of Biomedical Sciences
School of Veterinary Medicine, 100E
University of Pennsylvania
3850 Baltimore Avenue
Philadelphia, PA 19104-6009
Telephone: 215-898-8805
Fax: 215-898-0667
Email: volka@vet.upenn.edu


Appearance of spermatogonial stem cell cultures of rodent and non-rodent species. When grown in vitro, these stem cells from species separated by approximately 70 million years of evolution are remarkably similar in morphology. The mouse, rat and rabbit stem cells can be maintained in culture for longer than a year. However, stem cells of the baboon and human will grow for only about two months before disappearing. Nonetheless, the stem cells from these diverse species all require glial cell line-derived neurotrophic factor (GDNF) for their growth, indicating the extensive conservation of metabolic regulation in these stem cells. Experiments to understand the culture requirements of farm animals and primates are underway, including studies of genetic regulation of SSC self-renewal and spermatogenesis.

Spermatogonial stem cell cultures 

Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiol Rev. 2012 Apr; 92(2): 577-595. PubMed Central PMCID: PMC3970841.

Wu X., Goodyear SM, Abramowitz LA, Bartolomei MS, Tobias JW, Avarbock MR, Brinster RL. Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years. Human Reproduction. 2012 May; 27(5): 1249-1259. PubMed Central PMCID: PMC3329194.

Wu X., Goodyear SM, Tobias JW, Avarbock MR, Brinster RL. Spermatogonial stem cell self-renewal requires ETV5 mediated downstream activation of Brachyury in mice. Biol. Reprod. 2011 Dec; 85(6): 1114-1123. PubMed Central PMCID: PMC3223249. 

Niu Z, Goodyear SM, Rao S, Wu X, Tobias JW, Avarbock MR, Brinster RL. MicroRNA-21 Regulates the Self-Renewal of Mouse Spermatogonial Stem Cells. Proc. Natl. Acad. Sci USA. 2011 Aug 2; 108(31): 12740-12745. PubMed Central PMCID: PMC3150879.

Kubota H, Wu X, Goodyear SM, Avarbock MR, Brinster RL. Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties. FASEB J. 2011 Aug; 25(8): 2604-2614. PubMed Central PMCID: PMC3136339.

Oatley JM, Kaucher AV, Avarbock MR, Brinster RL. Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod. 2010 Sep; 83(3): 427-33. PubMed Central PMCID: PMC2924805.

Wu X, Oatley JM, Oatley MJ, Kaucher AV, Avarbock MR, Brinster RL. The POU domain transcription factor POU3F1 is an important intrinsic regulator of GDNF-induced survival and self-renewal of mouse spermatogonial stem cells. Biol Reprod. 2010 Jun; 82(6): 1103-1111. PubMed Central PMCID: PMC2874496.

Wu X, Schmidt JA, Avarbock MR, Tobias JW, Carlson CA, Kolon TF, Ginsberg JP, Brinster RL. Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules. Proc Natl Acad Sci USA. 2009 Dec 22; 106(51): 21672-7. PubMed Central PMCID: PMC2799876.


Oatley JM, Oatley MJ, Avarbock MR, Tobias JW, Brinster RL. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development. 2009 Apr; 136(7): 1191-9. PubMed Central PMCID: PMC2685936. 

Oatley JM and Brinster RL. Regulation of spermatogonial stem cell self-renewal in mammals. Annu. Rev. Cell Dev. Biol. 2008; 24: 263-286. PubMed Central PMCID: PMC4066667.

Oatley JM, Avarbock MR, Brinster RL. 
Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on SRC family kinase signaling. J. Biol. Chem. 2007 Aug 31; 282: 25842-25851. PubMed Central PMCID: PMC4083858.

Brinster RL. 
Male germline stem cells: From mice to men. Science. 2007 Apr 20; 316: 404-405. DOI: 10.1126/science.1137741; PubMed PMID: 17446391.

Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. 
Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc. Natl. Acad. Sci. USA. 2006 Jun 20; 103: 9524-9529. PubMed Central PMCID: PMC1480440.

Kubota H, Avarbock MR, Brinster RL. 
Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci USA. 2004 Nov 23; 101(47): 16489-94. PubMed Central PMCID: PMC534530.

Brinster RL. Germline stem cell transplantation and transgenesis. Science. 2002 Jun 21; 296: 2174-2176. DOI: 10.1126/science.1071607; PubMed PMID: 12077400.

 

For a full list of publications, please visit PubMed

Dr. Ralph Brinster, VMD/PhD, Penn Vet's Richard King Mellon Professor of Reproductive Physiology, is the first veterinarian and the eighth scientist from Penn to receive the National Medal of Science since its inception in 1963. Recognized as the father of transgenesis, Dr. Brinster's research on the manipulation of the mammalian germ line gave rise to this groundbreaking field.

Here are some news stories and magazine articles on Dr. Brinster and his work:

National Medal of Science

Dr. Brinster, President Barack Obama 

Here are additional resources to learn more about Dr. Brinster and the Brinster Laboratory of Reproductive Biology:

 Ralph L. Brinster

Personal

Military Service:                                                                               

1954-1956 Lieutenant, USAF

Office address:                                                                                

School of Veterinary Medicine
University of Pennsylvania
Philadelphia, Pennsylvania 19104

Telephone:  (215) 898-8805
Facsimile:   (215) 898-0667
Email: brinster@vet.upenn.edu
 

Education

1949-53       B.S., School of Agriculture, Rutgers University

1956-60       V.M.D., School of Veterinary Medicine, University of Pennsylvania

1960-64       Ph.D. (Physiology), Graduate School of Arts & Sciences, University of Pennsylvania

 

Additional Training

1960           Postdoctoral Fellow, Jackson Laboratory, Bar Harbor, Maine, Summer.

1962           Postdoctoral Fellow, Marine Biological Laboratory, Woods Hole, Massachusetts, Summer.

 

Appointments

1960-64      
Teaching Fellow, Department of Physiology, School of Medicine, University of Pennsylvania.

1964-65      
Instructor, Department of Physiology, School of Medicine, University of Pennsylvania.

1965-66      
Assistant Professor of Physiology, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA.

1966-70      
Associate Professor of Physiology, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania.

1968-83      
Program Director, Reproductive Physiology Training Program, School of Veterinary Medicine, University of Pennsylvania.

1969-84      
Program Director, Veterinary Medical Scientist Training Program, School of Veterinary Medicine, University of Pennsylvania.

1970-         
Professor of Physiology, School of Veterinary Medicine, University of Pennsylvania.

1970-         
Professor of Physiology, Graduate School of Arts & Sciences and Graduate Faculty, University of Pennsylvania.

1975-         
Richard King Mellon Professor of Reproductive Physiology, School of Veterinary Medicine and Graduate School, University of Pennsylvania.

1997-07      
Scientific Director, Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania

2007-08      
Co-Director, Institute for Regenerative Medicine, University of Pennsylvania.

 

Fellowships

1960-61      
American Veterinary Medical Association Fellow, Graduate School Arts & Sciences, University of Pennsylvania.

1961-64      
Pennsylvania Plan Scholar, Graduate School of Arts & Sciences, University of Pennsylvania.

 

Honors

New York Academy of Sciences Award in Biological and Medical Sciences, 1983.

Harvey Society Lecturer, 1984.

Member of the Institute of Medicine, National Academy of Sciences, 1986.

Fellow of the American Academy of Arts and Science, 1986.

Member of the National Academy of Sciences, 1987.

Honored (with L. Stevens) by an International Symposium at W. Alton Jones Cell Science Center (for pioneering work on development of the teratocarcinoma model and transgenic animals), 1987.

Fellow of the American Association for the Advancement of Science, 1989.

Distinguished Service Award of U. S. Department of Agriculture, 1989.

Invited Speaker at the Nobel Symposium on “Genetic Control of Embryonic Development”, 1991.

Pioneer Award from the International Embryo Transfer Society, 1992.

Juan March Foundation Lecture, Madrid, Spain, 1992.

Fellow of the American Academy of Microbiology, 1992.

Doctor Honoris Causa in Medicine, University of the Basque Country, Spain, 1994.

Charles-Léopold Mayer Prize (with R. Palmiter), The highest prize of the French Academy of Sciences (for development of transgenic animals), 1994.

Alumni Award of Merit, School of Veterinary Medicine, University of Pennsylvania, 1995.

First March of Dimes Prize in Developmental Biology (with B. Mintz) (for critical work in development of transgenic mice), 1996.

Carl Hartman Award, Society for the Study of Reproduction, 1997.

Bower Award and Prize for Achievement in Science, The Franklin Institute (for development of methods to transfer foreign genes into animals), 1997.

John Scott Award for Scientific Achievement, The City Trusts of Philadelphia, 1997.

Pioneer in Reproduction Research Award, The National Institute of Child Health & Human Development, National Institutes of Health, 1998.

Honored by a Special Festschrift Issue of the International Journal of Developmental Biology “Stem Cells and Transgenesis”, 1998.

George Hammel Cook Distinguished Alumni Award, Rutgers University, 1999.

Charlton Lecture, Tufts University School of Medicine, 2000.

Honorary Doctor of Science Degree, Rutgers, The State University of New Jersey, 2000.

Ernst W. Bertner Award, University of Texas M. D. Anderson Cancer Center (for research discoveries enabling development of transgenic animals), 2001.

Highly Cited Researcher (1980-2000), Designated by the Institute for Scientific Information, 2002.  About 1 in 1000 authors are in this category.

Selected for the Hall of Honor, National Institute of Child Health and Human Development, 2003, (1 of 15).

Wolf Prize in Medicine, Israel (with M. Capecchi and O. Smithies) (for introducing and modifying genes in mice), 2003.

Gairdner Foundation International Award, Canada (for pioneering discoveries in germ line modification in mammals), 2006.

National Medal of Science, USA (for fundamental contributions to the development of transgenic mice), 2010.                                         

International Society for Transgenic Technology Prize for outstanding contributions to the field of transgenic animals and stem cells, 2011.

Lifetime Achievement Award.  From the Alumni of the University of Pennsylvania School of Veterinary Medicine, 2012.

Career Excellence in Theriogenology Award.  From the Theriogenology Foundation on behalf of the American College of Theriogenologists and the Society for Theriogenology, 2012.