PICM01250 MilliporeMillicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm

BACKGROUND The initiation of meiosis is crucial to fertility. While extensive studies in rodents have enhanced our understanding of this process, studies in human fetal ovary are lacking. METHODS We used RT-PCR and immunohistochemistry to investigate expression of meiotic factors in human fetal ovaries from 6 to 15 weeks post fertilization (wpf) and developed an organ culture model to study the initiation of human meiosis. RESULTS We observed the first meiotic cells at 11 wpf, when STRA8, SPO11 and DMC1 are first expressed. In culture, meiosis initiation is observed in 10 and 11 wpf ovaries and meiosis is maintained by addition of fetal calf serum. Meiosis is stimulated, compared with control, by retinoic acid (RA) (P < 0.05). No major change occurred in mRNA for CYP26B1, the RA-degrading enzyme proposed to control the timing of meiosis in mice. We did, however, observe increased mRNA levels for ALDH1A1 in human ovary when meiosis began, and evidence for a requirement to synthesize RA and thus sustain meiosis. Indeed, ALDH inhibition by citral prevented the appearance of meiotic cells. Finally, 8 wpf ovaries (and earlier stages) were unable to initiate meiosis whatever the length of culture, even in the presence of RA and serum. However, when human germ cells from 8 wpf ovaries were placed in a mouse ovarian environment, some did initiate meiosis. CONCLUSIONS Our data indicate that meiosis initiation in the human ovary relies partially on RA, but that the progression and regulation of this process appears to differ in many aspects from that described in mice.

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages.

Organ culture systems of the central nervous system have proven to be useful tools for the study of development, differentiation, and degeneration. Some studies have been limited by the inability to maintain the cultures over an extended period. Here we describe an organ culture technique for the mouse retina. This method uses commercially available supplies and reproducible procedures to maintain healthy retinas with normal architecture for 4 weeks in vitro. The system is amenable to quantitative analysis. It can be used with both normal and retinal degeneration (rd) retinas to study of the role of various factors in photoreceptor degeneration in retinal cell fate determination and development.

Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.