Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

Abstract

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias)

“…The steady increase in cyst diameter during the spermatogonial stages is solely due to the proliferation of spermatogonia and Sertoli cells, at least for the first nine divisions. Careful analysis of DNA synthesis data in organ-culture experiments of immature rat testes similarly reveal an inverse relationship between germ-cell and Sertoli-cell proliferation, such that germ-cell DNA synthesis is low when Sertoli-cell DNA synthesis is elevated and vice versa (Schlatt et al 1999). Furthermore, FSH regulates both Sertoli-cell and differentiating spermatogonial DNA synthesis (Boitani et al 1995, Schlatt et al 1999.…”

“…Careful analysis of DNA synthesis data in organ-culture experiments of immature rat testes similarly reveal an inverse relationship between germ-cell and Sertoli-cell proliferation, such that germ-cell DNA synthesis is low when Sertoli-cell DNA synthesis is elevated and vice versa (Schlatt et al 1999). Furthermore, FSH regulates both Sertoli-cell and differentiating spermatogonial DNA synthesis (Boitani et al 1995, Schlatt et al 1999. By extension then, one explanation could be that seasonal alternating patterns in PCNA immunoreactivity in Sertoli and germ cells in sharks reflect variations in gonadotrophin secretion and/or changes in cell-type-specific responses to gonadotrophin secretion in these immature cysts.…”

“…Studies primarily in adult rodents indicate that, of the numerous regulatory mechanisms present within the testis, density-dependent (De Rooij 2001, Tadokoro et al 2002 and hormone-controlled (Meachem et al 1999, Allan et al 2004) mechanisms are two of the major physiological regulators that control these processes in an age-dependent and spermatogonial-stage-dependent manner. In addition, data obtained from organ cultures of immature rodent testes indicate that developing Sertoli cells influence the cellpopulation dynamics of gonocytes, and undifferentiated and differentiated spermatogonia, and their further maturation, depending on the age of the Sertoli cell (Boitani et al 1993, 1995, Zhou et al 1993, De Rooij & Van Dissel-Emiliani 1997, Schlatt et al 1999. Our knowledge of the inter-relationships between the different stages of spermatogonia and the various stages of Sertoli cell development in vivo is incomplete.…”

Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation

“…BrdU incorporation into testicular cells at 3, 9 and 18 dpp was detected by immunohistochemistry as previously described (Schlatt et al 1999), with minor modifications as described below (see Fig 1A). In brief, slides bearing paraffin-embedded tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (0·1 M, pH 6; 90-95 C for 10 min then room temperature for 20 min).…”

Effect of 17‐α‐ethynylestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis

“…Suitable methods to quantify cell proliferation are available (e.g. Thoolen 1990;Schlatt et al . 1999;Sakai 2002), and the biological effect of this synthetic estrogen addresses an interesting biological and environmental problem.…”