For more and more animal species, traditional approaches such as habitat protection and active population management are no longer sufficient because their populations have fallen below a critical level. The northern white rhinoceros is a prime example: in their natural habitat, numbers have dwindled over the past 30 years, rendering futile any attempts to launch a conservation initiative in their natural habitat in Northeast Congo or South Sudan. And with only two individuals left under human care (mother and daughter), conventional breeding programs are also no longer feasible. The situation is similarly precarious for the Sumatran rhinoceros: only a few dozen of Southeast Asia’s small, hair-covered rhinos remain – far too few for them to encounter one another regularly and reproduce. Even if all the remaining Sumatran rhinos were brought together at a breeding facility, the chances of offspring would be slim: owing to a lack of mating between the last individuals in their current fragmented and dispersed pockets, reproductive organs have degenerated in many cases, severely reducing their natural reproductive ability.
Last hope: the lab as an intermediate step
Strategies 1 and 2 – habitat protection and population management – are therefore not going to work for these ecologically important keystone species. For this reason, a consortium funded by the German Federal Ministry of Education and Research and based at the Leibniz-IZW started to develop a third strategy in 2019. This strategy is based on creating embryos by in vitro fertilization in the laboratory, rather than by fertilization in the female’s body. Egg cells and sperm are needed for this. In the case of the northern white rhinoceros, the “BioRescue” scientists, led by Professor Thomas Hildebrandt, were able to harvest egg cells from the female Fatu and fertilize them with cryopreserved and thawed sperm from deceased bulls in the laboratory – more than 20 embryos have been produced in this way. The embryos are to be carried out by surrogate southern white rhinoceroses – the only possibility to save the species.
This third strategy is also a possible means of stabilizing the Sumatran rhino population. With this species, the BioRescue team could as yet benefit from a larger pool of individuals capable of providing oocytes and sperm (gametes). But for both species, the limited availability and genetic variability of gametes are the bottleneck of the program. Besides Fatu’s oocytes, the program has access to frozen sperm from just four northern white rhino bulls – and some of those males are closely related to Fatu. This is detrimental in two respects: for one thing, the procedure involved in harvesting eggs is complicated and only a few oocytes can be collected; for another, this method limits the genetic diversity of any potential offspring. BioRescue seeks to eliminate this bottleneck using stem cell associated techniques (SCAT): the aim is to generate induced pluripotent stem cells (iPCS), primordial germ cells (PGCs) and, ultimately, artificial gametes from preserved tissue samples. This could increase the number of individuals in the founder group of a future population to 12 and permit the production of oocytes in much larger quantities. Stem cell specialists in the laboratories of Dr. Sebastian Diecke from Berlin’s Max Delbrück Center and of Professor Katsuhiko Hayashi from Osaka University, Japan, are therefore an essential part of the BioRescue consortium.
Uncharted research territory: creating rhino germ cells from stem cells
“In the long run, then, we will not only need a Strategy 3, but also a Strategy 3.2, called stem cell technology,” stated BioRescue Project Head Hildebrandt. “Adopting this route is the only way to achieve a sustainable viable rhino population in Central Africa and Southeast Asia.” However, enormous challenges need to be overcome for this to happen. After all, every single step – from using preserved rhino tissue samples to creating artificial oocytes or sperm – is uncharted territory for science. Together with its consortium partners, Leibniz-IZW was pleased to report major successes recently: at the Max Delbrück Center, BioRescue partners Dr. Vera Zywitza and Dr. Sebastian Diecke generated induced pluripotent stem cells from Sumatran rhino for the first time. The cells were from Malaysia’s last male Sumatran rhino, Kertam. The only surviving rhino species with hair has been considered extinct in Malaysia since 2019, following the death of Kertam and, just a few months later, female Iman. But this most recent success paves the way for the possibility of producing offspring from Kertam and other already deceased individuals.
In yet another recent success, the consortium proved that the next step in the stem cell strategy is also feasible: specialists at Osaka University led by Masafumi Hayashi and Katsuhiko Hayashi, in collaboration with Diecke’s laboratory and other BioRescue partners, succeeded in producing primordial germ cells from induced pluripotent stem cells from the northern white rhinoceros Nabire. This female lived in Dvůr Králové Zoo, Czech Republic, where she died in 2015 without any offspring. Primordial germ cells (PGCs) are the direct precursors of gametes, and the crucial link between stem cells and germ cells such as sperm and oocytes. To develop from stem cells, PGCs need a very specific environment in which signals from hormones or proteins trigger morphological and functional transformation. For the first time in large mammals, scientists have succeeded in creating such an environment in a culture system: The BioRescue team established culture systems for the southern white rhino, for which embryonic stem cells are available, and for the northern white rhino, for which they used iPCS derived from tissue samples. To succeed, they had to determine which signals needed to be introduced into the system, at what time, and in what order, so as to trigger the development to PGCs.
Embryos from artificial gametes also need surrogate females
As soon as the creation of artificial gametes is successful, this plan will be merged with Strategy 3.1, which BioRescue is carrying out using natural gametes: as with the oocytes harvested from Fatu and the sperm thawed from frozen samples, the artificially created eggs and sperm would be in vitro fertilized in the lab. The embryos produced in this way would be stored safely in liquid nitrogen until transfer to a surrogate female is possible. “These two paths we are taking are therefore not alternative routes, but are directly dependent on each other. We need both routes: advanced methods of assisted reproduction and stem cell technology,” remarked Hildebrandt. Traditional strategies such as active population management programs and habitat protection can only become effective again once populations of northern white rhinos and Sumatran rhinos have been created in this way.
The Sumatran rhinoceros was the topic of the “Echt oder Fake?” (Real or fake?) science show at the 30th anniversary FVB event at Berlin Science Week. Steven Seet, Head of Science Communication at the Leibniz-IZW, reported on the near-extinct rhino species, stating that is was possible to create stem cells and even precursors of eggs and sperm from skin cells. Is it really the case or, put differently: Is it true or fake? It is true!
Text: Jan Zwilling
The article was published in Verbundjournal 119 | 2022 with the focus on "30 years of FVB."
