Abstract
Although three-dimensional testicular cell cultures have been demonstrated to mimic the organization of the testis in vivo and support spermatogenesis, the optimal culture conditions and requirements remain unknown. Therefore, utilizing an established three-dimensional cell culture system that promotes differentiation of pre-meiotic murine male germ cells as far as elongated spermatids, the present study was designed to test the influence of different culture media on germ cell differentiation, Leydig cell functionality, and overall cell survival. Single-cell suspensions prepared from 7-day-old rat testes and containing all the different types of testicular cells were cultured for as long as 31 days, with or without stimulation by gonadotropins. Leydig cell functionality was assessed on the basis of testosterone production and the expression of steroidogenic genes. Gonadotropins promoted overall cell survival regardless of the culture medium employed. Of the various media examined, the most pronounced expression of Star and Tspo, genes related to steroidogenesis, as well as the greatest production of testosterone was attained with Dulbecco’s modified eagle medium + glutamine. Although direct promotion of germ cell maturation by the cell culture medium could not be observed, morphological evaluation in combination with immunohistochemical staining revealed unfavorable organization of tubules formed de novo in the three-dimensional culture, allowing differentiation to the stage of pachytene spermatocytes. Further differentiation could not be observed, probably due to migration of germ cells out of the cell colonies and the consequent lack of support from Sertoli cells. In conclusion, the observations reported here show that in three-dimensional cultures, containing all types of rat testicular cells, the nature of the medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation, due to the lack of proper organization of the Sertoli cells.
Highlights
Male infertility, a common disorder, is associated with a wide spectrum of spermatogenic failures, an increasing number of which are iatrogenic effects of clinical treatment [1]
INFLUENCE OF THE VARIOUS CULTURE MEDIA ON THE EXPRESSION OF STEROIDOGENIC GENES BY LEYDIG CELLS IN THREE-DIMENSIONAL CULTURES As assessed by quantitative PCR (qPCR), within 1 day of stimulation by gonadotropins the relative up-regulation of steroidogenic acute regulatory protein (Star) expression was fivefold with Dulbecco’s modified eagle medium (DMEM) + glutamine, threefold with F12, threefold with F12/non-essential amino acids (NEAA), twofold with F12/amino acids (AA), onefold with DMEM/F12, and fourfold with MEM (Figure 1B)
The major novel observations documented here are as follows: [1] the culture medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation in three-dimensional cultures; [2] rat germ cells migrating from the inner side to the outer side of the cell colonies suggest an unfavorable organization of tubules formed de novo in the three-dimensional culture; [3] undifferentiated rat spermatogonia differentiate up to the stage of pachytene spermatocytes in a similar time-period to the situation in vivo in three-dimensional cultures
Summary
A common disorder, is associated with a wide spectrum of spermatogenic failures, an increasing number of which are iatrogenic effects of clinical treatment [1]. Treatment of children with cancer, including radiotherapy and high-dose chemotherapy, can severely damage the immature gonads and lead to infertility later in life [2]. In three-dimensional cultures containing all murine testicular cells, testosterone production by the Leydig cells was enhanced in response to stimulation by hCG for as long as 16 days [6]. It remains to be determined whether similar Leydig cell function can be achieved with testicular cells from other species, including humans, under the same conditions
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