In March 2011, we reported successful in vitro spermatogenesis from murine spermatogonial stem cells to functional sperm.1 The method was a classical organ culture technique called gas-liquid interphase. Although the organ culture method is outdated, it was a reasonable strategy for successful in vitro spermatogenesis, because cultured tissue fragments are equipped with many constituents necessary for proper spermatogenesis, including somatic cells such as Sertoli and peritubular myoid cells, which form the seminiferous tubule structure.
However, the organ culture method itself did not yield promising results at the beginning of our study.2 We faced a difficulty in promoting spermatogenesis beyond pachytene-stage spermatocytes, as experienced in the 1960s.3 The solution was to use Knockout serum replacement (KSR; Gibco Life Technologies, Grand Island, NY, USA) in place of fetal bovine serum in the culture medium. KSR is a serum replacement commonly used in embryonic stem/induced pluripotent stem cell culture experiments. Now, we are trying to elucidate the precise factors that promote spermatogenesis in vitro. This is an important step in establishing human spermatogenesis in vitro.
The organ culture method has several advantages, possibly facilitating wider future applications. First, it is simple and easy to repeat with minimum basic culture equipment. Second, as it uses testis tissues per se, it is a half in vivo system causing less artificial stress to germ cells. For instance, the time course of spermatogenesis progression is the same as in vivo. This fact suggests that sperm produced in this system could have the same quality as in vivo. Third, cryopreservation of testis tissue was shown to be possible.1 Thus, it can be applicable, for instance, to cryopreserve testis tissues of pediatric cancer patients before chemo- and/or radiotherapies.
From my perspective, medical research on male infertility has stagnated over the past two decades, partly because of the introduction of the intracytoplasmic sperm injection (ICSI) technique in infertility clinics. What urologists can contribute to infertile couples is limited. The testicular sperm extraction (TESE) procedure, even though it is indispensable to obtain sperm for ICSI in azoospermic patients, is not a therapy, but merely rescuing sperm from unhealthy testicles. Now, it is desirable to treat testes so they can be induced to a healthy condition.
Along with such requests from many patients and from clinicians, I see a new movement in the area of male infertility clinics. It is a movement to treat the testes of patients. For instance, a trial has started to treat azoospermic patients with hormones to promote spermatogenesis before the TESE procedure. Some patients responded by showing successful sperm retrieval.4 Simultaneously, TESE samples can be a valuable resource to elucidate mechanisms of human spermatogenesis.5
The study of in vitro spermatogenesis, in a sense, is just starting, because its basis has now been established. It appears that male reproductive endocrinology is progressing to a new stage where the microenviromental hormonal milieu in testes can be studied in detail with organ culture methods. Combining such culture techniques with TESE samples and information on patients will show unknown mechanisms of spermatogenesis. Based on such research, new therapeutic modalities for male infertility will develop.