Two weeks ago, DNA Science covered the plight of the northern white rhino, suggesting assisted reproductive technologies (ARTs) that might preserve the genomes of the nearly-extinct subspecies. A paper published yesterday, in Nature Communication, reports creating embryos by injecting northern white rhino sperm nuclei into southern white rhino oocytes (unfertilized eggs).
The hybrid rhino embryos developed to a key early stage, the hollowed-ball blastocyst. If they can survive transfer to surrogate southern rhinos and continue developing, it would demonstrate that at least one route to salvaging the subspecies may be possible. But it might not be enough.
The blastocysts are “the first in vitro produced rhinoceros embryos ever,” said co-author Thomas Hildebrandt, of the German Leibniz Institute for Zoo and Wildlife Research. The northern white rhino population is down to just two infertile females (Najin and her daughter Fatu). The last male, Sudan, died in March. The southern subspecies is some 21,000 animals strong. Yet ironically, the genomes of northern animals, albeit based on a handful of samples, are more diverse.
The hybrid embryos, harboring one southern and one northern genome, are halfway to the goal of re-establishing a founding population of the dwindling subspecies. Mating the hybrids for several generations, serially selecting offspring with the highest percentages of northern DNA, could approach reconstituting the genome of the northern white rhinoceros, given time and luck. This is classical genetics sprung from an assisted reproductive technology.
Collecting Rhino Gametes
Even if a healthy, fertile male northern white rhino had been lumbering around, getting him to mount a southern rhino would hardly have been as simple as Gregor Mendel setting up pea crosses or me breeding fruit flies back in grad school. Yet collecting and merging gametes brings challenges too.
The procedure to create the hybrid embryos began with the delicately dubbed “transrectal ovum pick-up.”
Drugs stimulated the ovaries of southern females to pop out more mature oocytes, which the researchers plucked out with the animals under general anesthesia, using a patent-pending “rhinoceros-specific oocyte collection instrument.” The six-foot-long device is designed to easily reach a rhino’s ovaries through its rectum.
When an ultrasound scan revealed an oocyte about to emerge, the investigator introduced a double needle through the rhino’s intestinal wall and pierced the follicle. Out washed the oocyte, a little like trying to get the chocolate chips sunk to the bottom of a java chip frappuccino. The retrievals transpired not on the savannah of southern Africa, but in European zoos.
The process was labor-intensive. Eighteen retrievals from 314 ovarian follicles yielded 83 southern white rhino oocytes. The precious cells were shipped to the Avantea Laboratory of Reproductive Technologies in Cremona, Italy, for maturation and fertilization. But that required sperm.
While “good quality semen, fresh or frozen” is available from the abundant southern animals, northern white rhino semen comes from only three bulls. Many of the sperm couldn’t move their tails, and when injected into pig oocytes – a test of fertility – pooped out. And so the researchers turned to intracytoplasmic sperm injection (ICSI) to get sperm nuclei (genetic packages) into eggs, along with an electrical zap called assisted oocyte activation (AOA). ICSI and AOA are routine for horses and people.
The fertilized ova developed to the hollow-ball blastocyst stage in vitro. Cells on the interior face of the blastocyst, forming the all-important inner cell mass (ICM), divide into tissue layers that elaborate into organs. Meanwhile, the surrounding cells divide and form the associated membranes and structures.
Fertilization, or course, required exquisite timing. Upon arrival in Italy, the oocytes sat at body temperature for a little over a day, until the expulsion of a polar body from each indicated readiness for fertilization. (Polar body formation enables the forming female sex cell to reduce itself down to one genome copy). Thirty-two of the 83 oocytes made it this far. Of them, 19 were injected with sperm nuclei from southern white rhinos as a control just to see if it worked, while 13 received northern white rhino sperm nuclei.
Six of the 19 southern oocytes injected with southern sperm divided and 3 developed into blastocysts. Of the 13 oocytes injected with northern sperm, 6 divided and four reached blastocyst stage. “For the first time we had rhino blastocysts – an early stage of an embryo – developed in vitro, similarly to what we do routinely for cattle and horses,“ said co-author Cesare Galli.
Of the total seven embryos, four were the sought-after hybrids. Although they developed a bit slower and had smaller ICMs than the pure southern ones, the appearance of early embryos can be deceiving. Decrepit ones can be okay. Plus, the ICM cells resembled embryonic stem (ES) cells, which are actually cultured in a lab dish; they’re not embryo cells. And so the researchers collected and cultured ICM cells into ES cells, which indeed divided and gave rise to specialized cells representing all three layers of the embryo – a very promising finding that glimpsed the developmental potential of the northern-southern hybrid embryos.
Artificial Gametes and Embryos for the Future
The researchers froze some of the rhino ES cells, to possibly use them to generate “artificial” gametes, so-called because they don’t originate in ovaries or testes. The researchers also subjected various hybrid embryos or their parts to repeated freezing and thawing to test integrity, like I sometimes do with leftover lasagna.
The next step: implanting the embryos (fresh or frozen) into a uterus. Any uterus, for the genetic info comes in with the gametes.
ES cells aren’t the only route to fashioning artificial gametes. The researchers are also creating induced pluripotent stem (iPS) cells from banked somatic cells, such as skin fibroblasts, from several northern rhinos. If the iPS cells are successfully coaxed to form gametes, then IVF will be possible, speeding creation of pure northern embryos a different way.
Are ARTs in Conservation Biology Practical?
The experiments described in the new paper apply techniques and technologies that have been widely used in the livestock and human infertility industries for decades. In fact, the number of people conceived with IVF has just topped eight million. But using ARTs to rescue threatened and endangered species is far more nuanced and complex. Terri L. Roth and William F. Swanson, from the Center for Conservation and Research of Endangered Wildlife at the Cincinnati Zoo, point this out in a Comment accompanying the paper, “From petri dishes to politics – a multi-pronged approach is essential for saving endangered species.”
Even if ARTs can impregnate a few members of vanishingly small groups, the shrinking genetic diversity that emerges from population bottlenecks counters the birth and survival rate – a situation opposite that of the huge populations of domesticated animals whose genomes have been carefully selected and optimized for cranking out offspring. So it isn’t surprising that the track record of ARTs in conservation isn’t good. Roth and Swanson cite artificial insemination, one of the simplest ARTs, as an example. It works in dozens of mammal and bird species, but has helped “just three endangered mammals: the giant panda, black-footed ferret, and Asian elephant.”
What chance does the more complex creation of hybrid embryos, gestation in surrogates, and then selective breeding have of restoring the northern white rhino population to sustainable levels, with enough genetic diversity to overcome infertility? I think it unlikely, as do Roth and Swanson, who write that “impressive results in a Petri dish don’t easily translate into a herd of healthy offspring.” But might modifying genomes increase the odds of engineering healthy, fertile rhinos? Maybe.
But I’m uneasy with so much genetic and reproductive manipulation. Perhaps we need to acknowledge that in some cases saving a population that falls below a critical level of genetic diversity might not be possible, and that funds could be better used in conventional breeding programs, like the one for the Sumatran rhino.
I was once booed off a stage when, at the end of a talk on genetic modification, I said that extinction is sometimes an inevitability of circumstance and evolution. But that may indeed be the fate of the Northern white rhino. Sadly Nature doesn’t always find a way, even when we intervene.