The role of gonadotrophins in gonocyte transformation during minipuberty.

PURPOSE: Postnatal surge of gonadotrophins, Luteinizing hormone (LH) and Follicle-Stimulating hormone (FSH) known as minipuberty, is critical for gonocyte maturation into spermatogonial stem cells (SSC) in the testis. Gonadotrophins are essential for optimum fertility in men, but very little is known how they regulate germ cells during minipuberty. This study examined whether gonadotrophins play a role on gonocyte transformation in vivo. METHODS: Testes from hypogonadal (hpg) mice and their wild type (WT) littermates (n = 6/group) were weighed, and processed in paraffin at postnatal days (D) 0, 3, 6 and 9. Mouse VASA homologue (germ cell marker), anti-Müllerian hormone (Sertoli cell marker) antibodies and DAPI (nuclei marker) were used for immunofluorescence followed by confocal imaging. Germ cells on or off basement membrane (BM) and Sertoli cells/tubule were counted using Image J and analyzed with GraphPad. RESULTS: Comparing to WT littermates, there were significantly fewer germ cells on BM/tubule (p < 0.05) in D9 hpg mice, whereas there was no significant difference for germ cells off BM/tubule and Sertoli cells/tubule between littermates. However, testicular weight was significantly reduced in D3-D9 hpg mice comparing to WT littermates. CONCLUSION: Gonadotrophin deficiency reduced D9 germ cells on BM indicating impaired gonocyte transformation into SSC. This suggests that gonadotrophins may mediate gonocyte transformation during minipuberty.

Interplay between collagenase and undescended testes in Adamts16 knockout rats.

BACKGROUND: The inguinoscrotal stage of testicular descent is characterized by an increase in cell density and collagen fibers as the gubernaculum undergoes cell division and increases Extracellular Matrix (ECM) activity. Rats that lack the enzyme Adamts16, a known ECM proteinase, develop cryptorchidism postnatally and are infertile. Therefore, this study aims to investigate the link between the Adamts16 enzyme and congenital undescended testes (UDT) in Adamts16 knockout (KO) rats during postnatal development. METHODS: Formalin-fixed specimens of Wild-Type, Adamts16 heterozygous and Adamts16 homozygous KO rats post birth were sectioned and used for standard H&E histology and Masson's trichrome staining. A quantitative analysis on image J was performed to determine the intensity of collagen fibers within the inguinoscrotal fat pad (IFP) (n = 3 age/genotype). RESULTS: The migration of the gubernaculum within the Adamts16 heterozygous and Adamts16 KO rat was considerably disrupted. Furthermore, the Masson's trichrome staining demonstrated a significant increase in collagen fibers around the gubernaculum of rats that lacked Adamts16 enzyme at day 8. CONCLUSION: This study reports a failure of gubernacular migration leading to UDT in Adamts16 KO rats during development, suggesting that the expression of Adamts16 gene is critical for normal gubernacular migration through the breakdown of collagen fibers within the IFP.

Does the apoptosis pathway play a critical role in gonocyte transformation?

PURPOSE: Undescended Testes (UDT) are prevalent in 2%-5% of male infants and cause malignancy and infertility. During germ cell development, abnormal gonocytes usually undergo apoptosis. This process is believed to involve BAX (Bcl-2 Associated X) protein in clearing abnormal gonocytes which may fail in UDT, resulting in persisting gonocytes causing seminomas later in life. We aim to investigate the role of BAX in gonocyte apoptosis. MATERIALS AND METHODS: BAXKO (BAX-knockout) mice were back-crossed to OG2 mice (Oct4-promoter driving enhanced green fluorescent protein-eGFP) to produce BAXOG2 mice. Testes (wildtype-BAX+/+, heterozygous-BAX+/- and homozygous-BAX-/- mice, n = 6/group) on postnatal days 1, 3, 6, 9 were fixed and embedded in OCT for frozen sectioning. Sections were labeled with Anti-Müllerian Hormone (Sertoli cell marker), Mouse Vasa Homolog (germ cell marker) and DAPI (nucleus marker) and imaged using confocal microscopy. Oct4-GFP+ve germ cells, germ cells on/off the basement membrane and Sertoli cells were counted using ImageJ followed by data analysis with GraphPad. RESULTS: BAX-/-OG2 mice had significantly higher number of germ cells/tubule comparing to BAX+/+OG2 on day 9. There were Oct4-GFP+ve gonocyte-like germ cells that persisted in the center of the tubules in BAX-/-OG2 even after the completion of gonocyte transformation. This suggests that abnormal gonocytes in BAX-/-OG2 mice failed to undergo apoptosis and are allowed to persist. CONCLUSION: This study demonstrated that apoptosis is important in regulating germ cell migration and differentiation during gonocyte transformation in neonatal mice. In addition, inhibition of apoptosis results in persisting neonatal gonocytes which might become seminomas in patients with UDT.