Reproductive biology: In vitro sperm maturation

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Anticancer therapies can impair male fertility. Whereas men can opt to freeze their sperm before treatment, young boys don't produce mature sperm and so lack this choice. Work in mice offers hope for such patients. See Letter p.504

The blueprint for producing mature, functional spermatozoa in a laboratory dish — all the way from stem cells to flagellated sperm — has eluded reproductive biologists for decades. With a view to the eventual in vitro production of human sperm for clinical use, the main criterion of normal gamete function is the ability to support fertilization, with the subsequent development of normal offspring. Reporting on page 504 of this issue, Sato et al.1 meet this challenge in mice.

The process of spermatogenesis in mammals persists throughout almost all of adulthood. It starts with spermatogonial stem cells, which differentiate from type A spermatogonia into type B spermatogonia, and then into spermatocytes. The spermatocytes undergo meiotic cell division to form spermatids, and finally spermatozoa2. Complete maturation takes more than a month in most mammals.

Previous efforts to nurture sperm in vitro have either managed to recapitulate only parts of this complex differentiation process or failed to demonstrate the production of normal, fertile offspring. Although the organ-culture methods developed in the 1960s allowed germ cells to progress to meiosis3, until now no methods could support the entire process.

Sato et al.1 reasoned that the orchestration of cell-maturation signals during spermatogenesis should be achievable in a dish by providing nearly all of the cellular components found in the testes. To this end, they cultured testicular fragments to maintain the proper microenvironment for cell differentiation. This involved suspending fragments of immature testis on a semi-solid support, such as agarose, and partially bathing them in liquid. This organ-fragment culture system — referred to as the gas–liquid interface method — balances the delivery of nutrients from the culture medium to the maturing cells with the need for efficient gas exchange to maintain spermatogenesis for more than two months.

To visualize and score maturing germ cells under different conditions, the authors used a marker for spermatogenesis4: their mice were genetically engineered to express green fluorescent protein (GFP) under the control of regulatory elements for genes that are activated only when germ cells progress into meiosis and beyond. As expected, the tissues that they collected from the testes of newborn mice showed little or no baseline expression of GFP.

The authors had previously optimized4 various parameters involved in in vitro sperm formation, including temperature and the choice of basic ingredients for the medium. Notably, fetal bovine serum (FBS) seemed to be an important component, because its absence resulted in negligible maturation — as evidenced by the lack of the GFP signal.

In their present work, Sato et al.1 confirm the importance of FBS but, borrowing from the field of embryonic-stem-cell biology5, they find that an alternative to FBS known as knockout serum replacement (KSR) is even more effective. This finding is counter-intuitive, because KSR is commonly used to maintain stem cells in an undifferentiated state. A clue to the mechanism involved comes from the fact that the lipid-rich albumin component of KSR is itself highly effective in boosting differentiation.

The authors used in vitro fertilization (IVF) techniques to demonstrate the authenticity of the sperm collected from their cultures: they obtained male and female offspring that were themselves fertile.

The preservation of fertility is a major concern for patients requiring therapy, such as chemotherapy or radiation therapy, that can inadvertently destroy germ cells. In men, this problem can be mitigated by banking sperm before treatment. The solution is less straightforward in pre-pubescent boys. On the basis of pioneering work in animals by Brinster6 and others, the idea of transplanting cryopreserved spermatogonial stem cells is a reasonable strategy, although it has not yet been rigorously assessed in humans. Furthermore, the technology for the long-term culture and expansion of human spermatogonial stem cells has not been standardized, nor has the safety of the approach been tested.

Sato and colleagues' results suggest a viable alternative. In this scheme, boys would undergo testicular biopsy before chemotherapy or radiation therapy, to obtain tissue for cryopreservation (Fig. 1). If infertility occurs, the testicular fragments could be thawed and sperm obtained from organ culture for IVF. Such a protocol would bypass the need for surgical spermatogonial stem-cell transplantation.

Figure 1: Possible scenario for preserving fertility, based on the new work1.
Possible scenario for preserving fertility, based on the new work.

After a boy has been diagnosed with cancer, a biopsy would be performed to obtain testicular fragments for long-term cryopreservation. Later in life, when the individual wants to start a family, fertility would be assessed, and if he cannot produce functional sperm, the stored tissues could be thawed for organ culture. Sperm formed by this ex vivo method could then be used for in vitro fertilization (IVF).

It remains unclear whether the success of this system is due to signalling molecules released by the germ cells themselves, or to molecules released by the surrounding somatic (non-germ) cells, or to both. Nonetheless, the integrity of somatic cells, especially Sertoli cells, seems to be essential. However, this is not surprising, given that germ-cell maturation is known to depend on somatic-cell signals. In fact, even when embryonic stem cells have been used to produce germ cells in vitro7, signals contributed by differentiating testicular somatic cells in the culture seem to be required.

The exact nature of the external signals that enhance sperm maturation is not the only remaining mystery. It is also not known whether the resulting offspring — especially those produced from cryopreserved tissue — are generally healthy. Indeed, fertility of the offspring is just a crude indicator of whether gametes are 'normal'. Investigations should be made into whether the progeny Sato and co-workers generated by IVF are healthy in other ways (with respect to ageing, immune function, behaviour and so on).

As for the consequences of in vitro spermatogenesis at the molecular and cellular levels, previous data have indicated8 that adverse epigenetic effects occur when cells, especially gametes, are maintained in culture. Whether DNA repair, which is essential for spermatogenesis in vivo, functions normally in vitro is another concern. Subtle genetic or epigenetic changes could be pivotal for the well-being of subsequent generations. These caveats aside, the organ-culture approach1 represents a crucial experimental advance along the thorny path to the clinical use of sperm developed in vitro.


  1. Sato, T. et al. Nature 471, 504507 (2011).
  2. Russell, L. D., Ettlin, R. A., Hikim, A. P. S. & Clegg, E. D. Histological and Histopathological Evaluation of the Testis (Cache River Press, 1990).
  3. Staub, C. J. Androl. 22, 911926 (2001).
  4. Gohbara, A. et al. Biol. Reprod. 83, 261267 (2010).
  5. Price, P. J., Goldsborough, M. D. & Tilkins, M. L. International patent application: PCT/US98/00467 (1998).
  6. Brinster, R. L. & Zimmermann, J. W. Proc. Natl Acad. Sci. USA 91, 1129811302 (1994).
  7. Kee, K., Angeles, V. T., Flores, M., Nguyen, H. N. & Reijo Pera, R. A. Nature 462, 222225 (2009).
  8. Nayernia, K. et al. Dev. Cell 11, 125132 (2006).

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  1. Marco Seandel is in the Department of Surgery, Weill Cornell Medical College, New York, New York 10065, USA.

  2. Shahin Rafii is in the Department of Genetic Medicine and at the Ansary Stem Cell Institute, Weill Cornell Medical College, New York, New York 10065, USA.

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