A new model of development of the mammalian ovary and follicles

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.Katja Hummitzsch, Helen F. Irving-Rodgers, Nicholas Hatzirodos, Wendy Bonner, Laetitia Sabatier, Dieter P. Reinhardt, Yoshikazu Sado, Yoshifumi Ninomiya, Dagmar Wilhelm and Raymond J. Rodger

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2-3 months (Stage 1, 10 g), 4-5 months (Stage 2, 35 g), and 6-7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves

Investigation of Proliferative Activity in the Developing Human Tooth Using Ki-67 Immunostaining

Objective: The aim of this study was to investigate the proliferation of the developing human tooth germ and its surrounding tissues using Ki-67 immunostaining. Materials and Methods: Sections of mandibular dental arch tissues collected from 4 cadaveric human fetuses of 13, 16, 21 and 30 weeks of gestation were used. The immunoreactivity of Ki-67 in the tissue sections was assessed visually under a light microscope. Immunohistochemical controls were performed by replacing the primary antibody with phosphate-buffered saline or normal rabbit IgG. Results: The control sections did not display Ki-67 immunoactivity. Specimens of 13 weeks of gestation revealed intense Ki-67 immunostaining throughout the entire developing mandibular primary molars. At 16 weeks of gestation, immunostaining was observed in the inner enamel epithelium and dental papilla, in conjunction with the dental lamina showing decreased immunostaining. At 21 weeks, Ki-67 immunostaining was observed only in the inner enamel epithelium and dental papilla. The immunoreactivity of active ameloblasts and odontoblasts decreased, along with the proliferation capacity of the dental lamina. At 30 weeks, both enamel and dentin formation was observed along the cusped aspect of the tooth germ. Ameloblasts and odontoblasts were no longer immunoreactive in this region, while both types of cells were immunoreactive at the cervical regions of the crown. Dental lamina cells showed disintegration and were totally Ki-67-negative at 30 weeks of gestation. Conclusion: The Ki-67 immunoreactivity of the dental lamina decreased during intrauterine tooth development. Positive immunostaining was observed at specific sites in the enamel organ and dental papilla during the cap and bell stages. Copyright (C) 2007 S. Karger AG, Basel.WoSScopu