(as outlined and granted in 2008)
Oogenesis and spermatogenesis – two sides of the same medal
Oogenesis and spermatogenesis are complex processes by which haploid gametes are generated from primordial germ cells. Oocytes and sperm are two of the most differentiated cell types in our bodies, yet they can generate all cell types after fertilization. Owing to genomic imprinting, the two parental genomes are functionally different and normal development depends on both a paternal and a maternal contribution.
Whereas the basic principles of cell division and reduction of chromosomal content are highly similar between the two gametes, the timing of the commitment to enter meiosis shows a striking dichotomy in the male and female germ cells (for review see: Kimble & Page, 2007). In humans, a pool of germline stem cells that gives rise to oogonia and spermatogonia is generated prenatally. The mitotically dividing oogonia enter meiosis during fetal development and arrest at this stage, whereas meiosis in the male germline occurs at puberty. These sexually dimorphic differentiation properties result in the maintenance of a renewing stem cell population throughout the reproductive lifespan in the male but not in the female. This also has immediate consequences for therapeutical avenues of infertility treatments. While in men therapies can target the complete range of germ cells starting from the testicular stem cells, the spermatogonia, up to haploid spermatozoa, this treatment window in women is restricted to oocytes arrested in metaphase II up to the complete resumption of meiosis upon fertilization (Brinster, 2007).
Assisted Reproduction Techniques (ART)
Humans face the paradox situation of an exploding world population on the one hand, and on the other hand decreasing birth rates and negative population growth rates in developed countries such as Germany. This change can be attributed to a social climate in which couples prefer to have fewer children or none. In Germany, more than 20% of pregnant women are over 35 years, which is indicative of a socio-political climate that leads couples to postpone their decision to have children until a safe economical situation has been established. In addition, there is overwhelming evidence that environment, pollution and genetic alterations further reduce fertility. Taken together, these factors render 10-15% of all couples infertile. Simultaneously, major improvements have been achieved in the treatment of oncological patients, which led to higher life expectancy and a more frequent desire of these patients for having children, combined with a need for special fertility treatment options. These factors resulted in a strongly increased demand for Assisted Reproduction Techniques (ART). As of today, about 2% of children born in Germany are conceived through In Vitro Fertilization (IVF) with or without Intra-Cytoplasmic Sperm Injection (ICSI). Worldwide, more than 3 million children were born after ART. However, while there is a strong increase in the application of ART, scientifically the treatment procedure and options for IVF and ICSI and their risks are not thoroughly studied and mainly rely on descriptive studies. The efficiency of ART is still significantly lower than natural procreation. A prospective study on ICSI children revealed an increased risk of major congenital malformations and low birth weight compared to those born after spontaneous conception (Katalinic et al., 2004). There is concern that ovarian hyperstimulation and the culture of gametes and embryos for IVF/ICSI procedures might interfere with the complex epigenetic reprogramming of the DNA, which takes place during gametogenesis and early embryogenesis, and thus may increase the risk of imprinting defects leading to Beckwith-Wiedemann syndrome and Angelman syndrome. (Horsthemke and Ludwig, 2005).
Genetic alterations are transmitted in ART procedures, causing subfertility in progeny with unknown consequences for future generations. With respect to circumventing the severe impact of the hormonal stimulation regimen during ART on women and as an option for cancer patients, new avenues for fertility preservation by cryopreservation (vitrification) of oocytes, as well as in vitro maturation of oocytes (IVM), are being developed worldwide. Similar to IVF/ICSI, IVM was implemented into the clinical routine without knowing the basic principles and molecular mechanisms underlying germ cell maturation and the possible risks associated with incomplete maturation or overripeness.
Moreover, assisted reproduction techniques, such as the in vitro production of embryos and nuclear transfer cloning, play an increasingly important role in animal breeding, particularly for the genetic improvement of functional traits, e.g. metabolic stability, fertility and longevity. However, as in human, these techniques require knowledge-based optimization before they can be widely applied in breeding programs.
In vitro Gametogenesis
Recently, the potential of embryonic stem (ES) cells for the in vitro generation of gametes has been elegantly demonstrated (Hubner et al., 2003; Nayernia et al., 2006). Moreover, it has been shown that testes contain spermatogonial stem cells (SSCs) with unique features. Not only can these SSCs be matured by germ cell transplantation into recipient animals to yield haploid sperm, but they can also be transformed in vitro into multipotent adult germline stem cells capable of differentiating into the three germ layers ecto-, endo- and mesoderm (Guan et al., 2006). Thus, the testis is a unique source of adult stem cells, which are present at relatively high numbers and can be accessed easily by testicular biopsies. Such patient-specific testicular stem cells would offer promising therapeutic applications for infertile men.
These exiting studies are however limited by the fact that up to now, the basic mechanism for in vitro derivation of germ cells and their propagation and differentiation are poorly understood. Establishment of in vitro culture systems would allow to specifically studying the germ cell potential and their application in therapies. Oogonia or spermatogonia maintained in vitro could be targeted for genetic manipulation or directed to proliferate, undergo meiosis and complete gametogenesis. For example, the in vitro maturation of germ cells will have an important role in ART for patients suffering from a meiotic arrest during spermatogenesis by its potential to overcome the in vivo pre-meiotic block. A similar scenario could be envisaged for patients suffering from oncological treatments that are gonadotoxic. Such in vitro-cultured germ cells could i) increase by mitotic divisions the number of available SSCs, ii) be auto-transplanted to the testes to allow preservation of fertility after successful therapy, or iii) be further propagated to yield haploid sperm suitable for ICSI.
Concept and objectives
The Research Unit will explore and exploit the potential of germ cells. It combines stem cell research and reproductive medicine. Using modern cellular and molecular techniques (stem cell culture, 2D and 3D microscopy, methylation analysis, expression profiling and proteomics), it will investigate the development and the decline of the germ cell potential during in vivo and in vitro gametogenesis. A major focus will be the genetic and epigenetic determinants of the germ cell potential.
The projects will take advantage of the particular strengths of well-established animal models and clinical studies:
- Mouse (short generation time and excellent genome resources)
- Cow (mono-ovulatory mammal)
- Non-human primates (highly similar to humans, material easily available from tissues and developmental stages that are difficult to obtain in humans)
- Humans (patients with naturally occurring mutations that impair spermatogenesis and oogenesis; children conceived after in vitro maturation of oocytes
The Research Unit is composed of basic and clinical scientists, who work in gynecology, andrology, veterinary medicine, genetics and developmental biology. We expect that this multidisciplinary approach will not only advance our understanding of the germ cell, but also generate the basis for novel treatment protocols.
At short-term we expect to
- Identify genetic, epigenetic and biochemical factors involved in germ cell development, maturation and aging,
- Improve protocols for the therapeutic use of in vitro matured oocytes,
- Monitor the safety of in vitro maturation.
At long-term we expect to
- Delineate essential cellular and molecular pathways that define germ cell potential,
- Develop novel strategies to treat male and female infertility,
- Exploit the potential of spermatogonial stem cells for regenerative medicine.
Table 1: List of projects
Project 1: Role of structural genomic variation (copy number variants) in spermatogenetic failure.
F.Tüttelmann, B. Dworniczak; P. Wieacker (Münster)
Project 2: Spermatogonial stem cells and their potential.
W. Engel (Göttingen), U. Zechner (Mainz), W. Schulze (Hamburg)
Project 3: Derivation of pluripotent stem cells from juvenile marmoset (Callithrix jacchus) testes.
J. Gromoll, H. Schöler (Münster)
Project 4: Genetic analysis of Premature Ovarian Failure (POF syndrome).
P. Wieacker (Münster
Project 5: In vitro derivation and maturation of oocytes from murine embryonic stem cells.
H. Schöler, K. Hübner (Münster)
Project 6: Epigenetic status, proteome profile and health of in vitro grown and matured fresh or vitrified mouse and human oocytes.
U. Eichenlaub-Ritter (Bielefeld), G. Griesinger (Lübeck), T. Haaf (Mainz), G.J. Arnold (München)
Project 7: Epigenetic analysis of imprinting in the bovine oocyte.
H. Niemann (Mariensee), C. Wrenzycki (Hannover), T. Haaf (Mainz)
Project 8: Oocyte maturation and developmental competence at different life cycle stages: structural, molecular and functional analyses in the bovine model.
F. Habermann, G.J. Arnold, E. Wolf (München)
Project 9: Follow-up of pregnancies established after in vitro maturation and consecutive IVF.
M. Ludwig (Hamburg), T. Strowitzki (Heidelberg), A. Katalinic (Lübeck), M. von Wolff (Heidelberg), S. von Otte (Lübeck), K. Diedrich (Lübeck), B. Horsthemke (Essen)
Project M: Management of the Research Unit.
J. Gromoll (Münster)
The Research Unit will meet once a year to discuss the results of the projects. The meeting will be open to postdocs and graduate students who work on the subprojects to allow informal exchange of protocol and data details, discussions on scientific and technical problems, and will broaden their horizon. We will also invite two or three foreign speakers to reflect our progress in the light of international developments in the field.
Integration and promotion of young scientists
Junior Research Group
We will provide lab space in the participating departments in Münster for a junior research group funded by the Emmy Noether Programme by the DFG. We aim at recruiting an outstanding young post-doc currently working abroad in order to broaden the basis of reproductive biology and medicine in Germany. For example, a research group on piwi-interacting RNAs (a group of small RNAs that interfere with transcription of genes), which play an important role in the germline, would be highly welcome, because this topic is currently not being investigated in the field of reproductive biology/medicine in Germany. Yet, it is considered to be one of the most prominent topics in future reproductive research (for a review on the impact of piwis on the germline please see O’Donnell K and Boeke JD, 2007). The junior group will be closely associated with the Research Unit.
Recruitment of students
Central to our application is the wish to attract young scientists to the field of our research. We will offer students the possibility to visit the participating laboratories for a one-week course that will give them a first glimpse into reproduction research. In addition, the courses will facilitate the recruitment of bright candidates for Bachelor, Master, MD and PhD degrees. The laboratory visit program will of course also apply to PhD students and postdocs funded by the Research Unit in order to perform collaborative experiments or to learn specific techniques.
- Brinster R (2007) Male germline stem cells: From mice to men. Science 316:404-407
- Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G (2006) Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 440:1199-203
- Horsthemke B, Ludwig M (2005) Assisted reproduction – the epigenetic perspective. Human Reprod Update 11:473-482
- Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Scholer HR (2003) Derivation of oocytes from mouse embryonic stem cells. Science 300:1251-1256
- Katalinic A, Rosch C, Ludwig M; German ICSI Follow-Up Study Group (2004) Pregnancy course and outcome after intracytoplasmic sperm injection: a controlled, prospective cohort study. Fertil Steril 81:1604-1616.
- Kimble J, Page DC (2007) The mysteries of sexual identity: The germ cell’s perspective. Science 316:400-403
- Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N, Dev A, Wulf G, Ehrmann IE, Elliott DJ, Okpanyi V, Zechner U, Haaf T, Meinhardt A, Engel W (2006) In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 11:125-132.
- O’Donnell KA, Boeke JF (2007) Mighty piwis defend the germline against genome intruders. Cell 129: 37-44