In the adult male reproductive tract, the epididymis is of crucial importance for fertility. Only spermatozoa that have passed through the six-metre-long epididymal duct in about ten days acquire their fertilising ability. The smooth muscle cells of the duct play an important role in the transport of the still immotile spermatozoa of the testes. In preliminary work, our research group was able to demonstrate muscle cell contractions in the rat epididymis immediately after birth resulting in the directed transport of desquamated epithelial cells in the lumen of the epididymis. Contractions could be accelerated by adrenergic signalling pathways and slowed down by the cGMP pathway. Such a transport process requires a differentiated smooth muscle layer, which has been examined in rodents, but not yet in humans. Fetal human epididymal samples were available starting at the 15th week of pregnancy. Analyses of these samples were expected to provide information on the expression of smooth muscle cell markers allowing to draw conclusions of cell differentiation. Various structural proteins, including smooth muscle actin (SMA), calponin and myosin heavy chain (MYH), were examined, along with the cGMP-dependent protein kinase I (PKG I), as part of the relaxing signalling pathway in muscle cells. In immunohistochemical analyses, SMA, a marker that is expressed independently of the differentiation state of the cells, was detected in smooth muscle cells even in the earliest available sample (15th week of pregnancy). Beginning at the 21st week of pregnancy, calponin indicated progressive differentiation of the smooth muscle cells. Immunofluorescence staining from this week onwards also confirmed a co-localisation of SMA and calponin in the smooth muscle layer. Later in development, MYH was observed by immunohistochemical examinations as a further structural protein from the 25th week of pregnancy onwards. Moreover, PKG I was first seen around the 28th week of pregnancy. All tested muscle cell markers were also detected in each available postnatal human epididymis (15d, 24d, 27d) and in adult samples. Overall, in the epididymis minor muscle differentiation was observed at the beginning of development compared to the end of the fetal period, in postnatal and adult tissue. Full differentiation could already be observed in the fetal epididymal duct from the 28th week of gestation, suggesting that a transport function is likely from this point on and at all postnatal examination times. The less differentiated muscle cells before might be involved in the development of a 3D epididymal structure in the context of coiling. Interestingly, the expression and thus the differentiation state of the smooth muscle cells in the epididymis correlate with the testosterone level, suggesting a hormonal influence. The results obtained in this work provide new insights into the development and function of the epididymis and could be of great relevance for the time of developmental disorders and infertility in men.
