World of Reproductive Biology

BOR–Papers in Press published 3 October 2012 DOI: 10.1095/biolreprod.112.105338 A study in mice points to a new approach to preserving fertility in female cancer patients. Chemotherapy can wreak havoc on DNA. The drugs can damage the DNA of the oocyte pool, resulting in the induction of a cell-death program and culling of primordial oocytes, which provide a lifetime's supply of eggs. A consequence of cancer therapy in women is often reduction of the ovarian reserve, resulting in impaired fertility and early menopause. Approaches to fertility preservation currently rely mainly on freezing oocytes or ovarian tissue prior to chemotherapy. But reproductive biologists have been seeking ways to protect oocytes from the effects of the chemotherapeutic agents by pharmacological means. The new study edges researchers closer to this goal. In this investigation, Jeffrey Kerr, Karla Hutt, Ewa Michalak, and colleagues took a close look at the triggers for oocyte death in response to DNA-damaging agents. Consistent with previous results, they found that DNA damage induces the activity of the transcriptional regulator TAp63, a homolog of Trp53—also known as p53, the famed “guardian of the genome.” The researchers also showed that TAp63 induced the expression of two factors that promote apoptosis, Puma and Noxa (both are members of the BH3-only pro-apoptotic subgroup of the wider BCL-2 family). Crucially, they showed that the primordial oocytes in mice lacking Puma or both Puma and Noxa were protected from apoptosis induced by DNA damage. These females could also produce apparently healthy offspring with no overt deformities and normal fertility. The mice lacking these genes also seemed to have active DNA repair pathways: A protein involved in double-stranded break repair (γ-H2AX) accumulated on damaged oocyte DNA in the oocytes of these mice, but was largely absent after five days, suggesting that the repair protein had done its job. It's unclear whether the offspring suffer from damage to their DNA that might yield more subtle effects, and the researchers acknowledge the need for longer-term follow-up studies. If the same mechanism operates in humans, drugs that block PUMA and NOXA may be useful for preventing infertility in female cancer patients and could possibly even fend off menopause in healthy women. Kerr JB, Hutt KJ, Michalak EM, Cook M, Vandenberg CJ, Liew SH, Bouillet P, Mills A, Scott CL, Findlay JK, Strasser A. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa. Mol Cell 2012; 48(3):343–352. BOR–Papers in Press published 10 October 2012 DOI:10.1095/biolreprod.112.105551 Mouse stem cells have been coaxed into producing oocytes, Japanese researchers report. Last year, Mitinori Saitou and colleagues showed that, using either embryonic stem cells or induced pluripotent stem cells, they could create viable precursors of sperm in a petri dish. These cells developed into functional sperm after transplantation into male mice. The researchers have now adjusted their protocol to produce viable oocytes by incubating them with fetal ovarian cells and transplanting them into adult females. Using a brew of various growth factors, the researchers first generated cells with the properties of primordial germ cells. To turn these primordial cells into oocytes, the researchers cultured them with immature somatic cells from fetal ovaries. The cultured cells together formed an ovary-like structure that was transplanted into nude mice, where further development ensued. The germ cells from these ovaries were able to initiate meiosis and had other characteristics of oocytes, although they deviated from their in vivo-produced counterparts in some ways. For instance, when isolated from the reconstituted ovaries, these oocytes tended to shed the specialized granulosa cells that are normally connected to the oocytes in a cumulus-oocyte complex. Another interesting difference was that the follicles developed in synchrony, in contrast to the usual pattern of follicle development, where most of the oocytes remain in reserve in primordial follicles. Despite these differences, the oocytes were functional after transplantation into an adult female. In vitro fertilization with these oocytes was successful, resulting in live offspring with no apparent abnormalities. These findings may lead to methods for producing oocytes in vitro for preservation of endangered species or to treat infertility, for instance in women undergoing cancer therapy. The results may have the most immediate impact on basic research, providing a source of oocytes and a way to study their development. Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, Saitou M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 2012; 338(6109):971–975. BOR–Papers in Press published 17 October 2012 DOI:10.1095/biolreprod.112.105668 During pregnancy, the fetus is protected from immune attack by a shield of protective immune cells called regulatory T cells. These cells put the damper on the immune system, which would otherwise treat the fetus, bristling with foreign antigens from the father, as an alien being. New findings show that regulatory T cells remain at the ready in subsequent pregnancies, rapidly reaccumulating when exposed to fetal antigens present in the first pregnancy. The result is an even more powerful immune shield during the second pregnancy. Previous studies have shown that regulatory T cells are induced during pregnancy. Studies in mice have shown that without these cells, the immune system attacks the fetus, leading to its reabsorption. Jared Rowe et al. took a closer look at regulatory T cells during pregnancy, quantifying their response to a specific antigen in the fetus. They used male mice engineered to express a specific, well-characterized antigen and mated those mice with females lacking the antigen. Next, using standard immunological research tools, they analyzed the mother's response to this antigen. The researchers found that pregnancy resulted in a more than 100-fold increase in regulatory T cells specific for the antigen. These regulatory T cells were sustained at enriched levels after delivery, ready to be deployed in a second pregnancy. During the second pregnancy, these cells rapidly expanded if the fetus expressed the same original antigen. Moreover, the fetus in the second pregnancy was better shielded from fetal absorption in experiments partially ablating regulatory T cells. The findings are in sync with recent immunological studies showing that regulatory T cells maintain a memory of self-antigens and are primed to respond rapidly to them. The findings also might provide a mechanistic explanation for patterns of risk. Women are at reduced risk for pre-eclampsia and other pregnancy complications associated with disrupted fetal tolerance in their second pregnancy, and these protective effects are most pronounced if subsequent pregnancy is with the same partner. Perhaps this is because pregnancy-induced regulatory T cells are prepared to more efficiently protect the fetus in secondary pregnancy from immune attack by the mother. Rowe JH, Ertelt JM, Xin L, Way SS. Pregnancy imprints regulatory memory that sustains anergy to fetal antigen. Nature 2012; 490(7418):102–106. BOR–Papers in Press published 24 October 2012 DOI:10.1095/biolreprod.112.105767 In essentially all animal species, mitochondria are inherited from the mother, who provides a genetically uniform population of these organelles in the egg. Experiments by Mark Sharpley, Christine Marciniak, and colleagues now suggest an explanation for the evolution of this phenomenon: mice with two genetically distinct populations of normally-functioning mitochondria suffer from reduced activity, cognitive impairment, and other afflictions. To create mice with chimeric mitochondrial populations, the researchers fused an embryonic stem cell containing mitochondria from one strain of mice with the cytoplasm of a cell containing another mitochondrial type. Mice containing uniform populations of either organelle were healthy. The resulting cells were cultured and then incorporated into mouse blastocysts to yield mice with cells containing both mitochondrial populations. The nuclear genotype of the mice was uniform. The researchers bred the mice for over ten generations, assessing the proportion of each type of mitochondria and examining the health of the mice. They found that over generations, the mitochondrial genome tended toward homoplasmy, with one to the two mitochondrial types persisting with greater frequency due to selection in the female germline. The researchers also found that mice with 50:50 mixtures of the two mitochondrial types had strikingly poor health. For instance, those mice were less active, ate less, and showed impaired learning, taking four times longer to escape from a maze than siblings containing only one mitochondrial population. They also reacted differently to stress. These health issues did not occur if the proportion of one of the mitochondrial types dipped below 10 percent. The phenotype suggests that the mice had alterations in energy usage, for instance responding poorly when extra energy was required in the brain. But the mechanistic basis for the effects is not clear. Mitochondria, which fuse constantly inside of cells, encode for components of the machinery involved in oxidative phosphorylation. Perhaps mixing the two mitochondrial DNAs and their protein products reduces the efficiency of oxidative phosphorylation. Or maybe a mixture of mitochondria perturbs a regulatory feedback loop between the organelle and its cellular host that regulates energy states; perhaps the number of mitochondria in a cell is sensitive to the mitochondrial genotype. In a commentary associated with this report, Nick Lane speculates that the need for uniparental inheritance of mitochondria may have led to the evolution of male and female sexes. “There is no obvious reason why there should be two distinct sexes,” he says. Perhaps mitochondria provided the evolutionary impetus. Sharpley MS, Marciniak C, Eckel-Mahan K, McManus M, Crimi M, Waymire K, Lin CS, Masubuchi S, Friend N, Koike M, Chalkia D, Macgregor G, et al. Heteroplasmy of mouse mtDNA is genetically unstable and results in altered behavior and cognition. Cell 2012; 151(2):333–343. Lane N. The problem with mixing mitochondria. Cell 2012; 151(2):246–248. BOR–Papers in Press published 31 October 2012 DOI:10.1095/biolreprod.112.105940 A technique involving transfer of DNA between human oocytes now brings researchers closer to the clinical goal of preventing the transfer of mitochondrial disease from mother to child. Mitochondrial diseases can have a range of symptoms that often affect the functioning of organs with high energy requirements, such as the heart or brain. About one in every 5,000 to 10,000 babies are born with aberrant mitochondria inherited from their mother via the oocyte. About three years ago, Shoukhrat Mitalipov and colleagues performed a DNA-swapping experiment in monkeys with genetically distinct mitochondrial and nuclear DNA. They removed the nuclear chromosomes from the oocyte of a rhesus macaque and transplanted them into the cytoplasm of a macaque with a distinct nuclear haplotype that had its nuclear chromosomes removed. They successfully fertilized these oocytes, which yielded live offspring carrying different nuclear and mitochondrial haplotypes. In their current Nature report, the team shows that after three years, these monkeys remain healthy and their mitochondrial function does not seem to be impaired. In the current study, the researchers also performed the chromosome-swapping procedure on 65 human oocytes from seven healthy donors. About 73 percent of the eggs were fertilized successfully, and 62 percent of those developed up to the blastocyst stage, when they were used to derive embryonic stem cells. Further experiments showed that these embryonic stem cells contained less than one-percent carryover mitochondrial DNA. The findings suggest that the technique might be successfully applied to the oocytes of women with mitochondrial disorders, but the new study also revealed some concerns. About half of the manipulated oocytes had abnormal fertilization, as determined by the presence of an extra pronucleus, which may indicate abnormal chromosome number. The researchers say that separating normal and abnormal embryos could be accomplished by morphological evaluation after fertilization and detection of abnormal pronucleus formation. In the future, researchers may hone the technique to minimize aberrant fertilization. Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, Ma H, Gutierrez NM, Tippner-Hedges R, Kang E, Lee HS, Ramsey C, Masterson K, et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature 2012; (in press). Published online ahead of print 24 October 2012; DOI 10.1038/nature11647.