Gonocyte Migration Research Articles

Postnatal Germ Cell Development during Mini-Puberty in the Mouse Does Not Require Androgen Receptor: Implications for Managing Cryptorchidism

Undescended testis leads to infertility and malignancy resulting from aberrant germ cell development. Androgens are proposed to control early germ cell development during the transient postnatal surge of gonadotropins and androgen, known as mini-puberty. We assessed the effect of androgen receptor on perinatal germ cell development in mice. Testes from androgen receptor knockout mice and wild-type littermates (3 to 4 per group) were collected at embryonic day 17 and postnatal days 0 (birth), 2, 4, 6, 8 and 10 for immunohistochemical analysis. Antibodies against mouse VASA homologue (germ cell marker), antimüllerian hormone (Sertoli cell marker), Ki67 (proliferating cell marker) and DAPI (nuclei) were used and visualized by confocal microscopy. Number of germ cells per tubule, germ cells on the tubular basement membrane and Sertoli cells per tubule, and percentage of proliferating germ cells (Ki67(+)) per tubule and germ cells (Ki67(+)) on the basement membrane on confocal images were counted using Image J, version 1.44 (http://imagej.nih.gov/ij/). Data were analyzed using nonparametric one-way ANOVA with GraphPad Prism® 5.02 software. In wild-type and androgen receptor knockout testes germ cells per tubule decreased from embryonic day 17 to postnatal day 2, then increased normally. Number of mouse VASA homologue positive germ cells per tubule and germ cells on the basement membrane were similar in androgen receptor knockout and wild-type testes (p > 0.05) at each age, and percentages of proliferating germ cells (Ki67(+)) per tubule and proliferating germ cells on the basement membrane were similar at each age (p > 0.05). Androgen receptors are not required for gonocyte migration from the center of the testicular tubules to the basement membrane and transformation into spermatogonia stem cells up to day 10 in androgen receptor knockout mice. Identifying nonandrogenic factors might improve the fertility potential of boys with undescended testis who are undergoing orchiopexy.

Is matrix metalloproteinase required in postnatal testicular tubules for germ cell maturation?

Background/AimCryptorchidism may cause infertility by failed transformation of neonatal gonocytes into adult dark spermatogonia, the putative stem cells for spermatogenesis. Gonocytes migrate centrifugally to the tubular basement membrane to become adult dark spermatogonia. Regulation of this transformation remains unknown. We aimed to investigate neonatal rodent testis matrix metalloproteinase (MMP) production to see whether MMPs loosen extracellular matrix between Sertoli cells to facilitate gonocyte movement. MethodsSprague-Dawley rat testes (n = 4-6 per group) were collected at embryonic day 19 (E19) and postnatal (P) days P0 to 10 for immunohistochemistry. Immunofluorescent confocal images were captured for presence of membrane type 1 MMP (MT1-MMP), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 2 (TIMP2), mouse VASA homologue, anti-Müllerian hormone, and androgen receptor in tissue sections. Testicular proteins were analyzed by immunoblotting. ResultsMembrane type 1 MMP was strongly present in gonocytes at E19 then decreased, whereas it increased in testicular somatic cells from P0 to P10. Testicular protein levels of MT1-MMP, MMP2, and androgen receptor were constant from E19 to P10. Anti-Müllerian hormone protein sharply decreased after P2, whereas TIMP2 gradually increased from E19 to P10. Gonocytes migrated to basement membrane at P2 to P6. ConclusionMembrane type 1 MMP, MMP2, and TIMP2 were present in testis from E19 to P10 during gonocyte migration and transformation into spermatogenic stem cells. Increased knowledge about germ cell development may aid efforts to improve fertility in cryptorchidism.

233. A role for activin/inhibin in mouse gonocyte relocation and proliferation

Gonocytes in the testis resume proliferation after birth and relocate to contact the basement membrane of the seminiferous cords where they become spermatogonia. A previous in vitro study indicated that activin can increase gonocyte numbers on day 3 postpartum (dpp) rat testis, while the activin antagonist, follistatin, together with FSH, increased the number of spermatogonia (Meehan et al. 2000). The aim of this study was to understand how FSH, activin and inhibin, a potent activin antagonist, interact to influence gonocyte proliferation and relocation in the newborn mouse testis using in vivo and in vitro approaches. Two mouse models were analysed, the inhibin α knock out (inh a −/−) mouse and the InhbaBK mouse. The Inh a −/− mouse lacks inhibin, and thus activin acts unopposed by its most potent antagonist (Matzuk et al. 1992). The InhbaBK mouse has the Inhbb allele inserted into the Inhba locus, thus directing the expression of the less bioactive activin βB, in the spatiotemporal pattern of activin βA (Brown et al. 2000). In addition, an in vitro model was developed in which 1dpp wild type testis fragments were cultured in hanging drops for 24 h with the addition of combinations of activin, inhibin and FSH. Gonocyte proliferation in inh a −/− was assessed using proliferating cell nuclear antigen (PCNA). A significant increase in germ cell proliferation and relocation to the basement membrane was measured in 0dpp inh a −/−, while no difference was observed at 4dpp. The opposite was observed in InhbaBK mice, with reduced gonocyte migration in mutant animals at 0dpp. In vitro, inhibin seemed to inhibit proliferation and reduce the percentage of relocated gonocytes while FSH showed a tendency for the opposite effect on gonocyte migration. These findings show that inhibin levels affect germ cell development during early postnatal development in mouse testis influencing both cell maturation and proliferation. (1) Meehan T, Schlatt S, O'Bryan MK, de Kretser DM, Loveland KL. Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Dev Biol. 2000 Apr 15;220(2):225–37. (2) Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature. 1992 Nov 26;360(6402):313–9. (3) Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues the Inhba null phenotype and reveals new activin functions. Nat Genet. 2000 Aug;25(4):453–7.

Spermatogenesis in immature mammals.

Mammalian spermatogenesis has been studied extensively as a prime theme of male reproductive biology, especially for germ cell production, fertilization and development. Investigation of spermatogenesis has provided us with the opportunity to both study the male germ line stem cells and generate the transgenic animals. Spermatogenesis is conducted in the seminiferous tubules, which end in the rete testis. The organization of spermatogenesis means that the spermatogonia are uniformly distributed around the seminiferous tubules. The pubertal establishment and mature maintenance of spermatogenesis requires precursor cells. In bull testes at 4weeks postnatal, gonocyte migration occurs and differentiated spermatogonia are recognized after 8weeks. Within the period of 4-8weeks of age, spermatogonial stem cell conversion and niche formation must occur. Spermatogonial stem cells are the only cells that can undergo self-renewal in spermatogenesis. Spermatogonial stem cell transplantation can potentially contribute to studies of gene expression during spermatogenesis and provide genetic progress in domestic animals. Bull spermatogonial stem cells have been demonstrated to be capable of colonizing recipient mouse seminiferous tubules. (Reprod Med Biol 2007; 6: 139-149).

Gonocyte-Sertoli cell interactions during development of the neonatal rodent testis

During neonatal testicular development in the rat, events critical for subsequent germ cell development occur that set the stage for fertility later in life. Some gonocytes resume mitotic activity and/or migrate to the surrounding basal lamina, and use of a carefully defined Sertoli cell-gonocyte coculture system indicates that these crucial events occur without added factors or hormones and are hence likely to depend on interaction with adjacent Sertoli cells. Coupling of the Kit receptor protein on gonocytes to stem cell factor from Sertoli cells is vital for successful migration by gonocytes, as antagonism of the former suppresses and addition of the latter stimulates gonocyte migration. During the neonatal period, intercellular adhesion is modified in a developmental manner such that neural cell adhesion molecule (NCAM) is the main adhesive molecule expressed and functioning at birth, with a progressive decline as development proceeds. This decline in NCAM expression is supported by the addition of exogenous 3,3',5-triiodothyronine in vitro, and because this factor is recognized as supporting Sertoli cell differentiation, it seems likely that changing intercellular adhesion is a function of progressive development of Sertoli cells. Other avenues whereby maturing testicular cells influence each other doubtless exist, including secretion of growth factors and other peptides and developmentally important changes in the makeup of the extracellular matrix, which Sertoli cells and gonocytes contact. Continued investigation in these areas will be very valuable in enlarging our understanding of how neonatal testicular development provides the basis for successful spermatogenesis.

Use of in vitro systems to study male germ cell development in neonatal rats

The aim of this review is to summarize ways in which in vitro approaches have allowed us to investigate several aspects of gametogenesis in the male. In our laboratory, we have established both organ culture and cell co-culture methodologies and applied them to questions focused on cellular and molecular events important for development of primitive spermatogonia, or gonocytes, in testes of neonatal rats. We have described their postnatal reinitiation of mitosis and their migration to the basal lamina in anticipation of basal compartment formation and, through use of these in vitro systems, we have identified several mechanisms regulating these processes. These include matrix influence on mitosis and migration, adhesive mechanisms active between gonocytes and Sertoli cells, and involvement of the Kit receptor on germ cells and its ligand from Sertoli cells in supporting gonocyte migration, as described below.

Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro.

Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position crucial for their survival. These cells express the c-kit gene from the day of birth through Day 5 in vivo and develop the ability to migrate in Sertoli cell-gonocyte cocultures. In this study, we asked whether c-kit expression and synthesis of Kit protein are required for pseudopod production by gonocytes in vitro. To determine whether gonocyte migration in vitro is invariably accompanied by c-kit expression, we quantified percentages of gonocytes expressing c-kit with increasing time in vitro and correlated these data with pseudopod development by individual cells. We also determined the effect of exposure to Kit antibodies on gonocyte migration in vitro, and, conversely, asked whether addition of exogenous stem cell factor (SCF), the Kit ligand, stimulates pseudopod development. We found that 1) increasing numbers of gonocytes express c-kit with increasing time in vitro; 2) once these cells begin migrating in vitro, the appearance of a pseudopod on a gonocyte is absolutely correlated with kit expression by that cell; 3) incubating cocultures with Kit antibodies significantly reduces the number of cells with pseudopods, without any detectable decrease in numbers of gonocytes; and 4) addition of exogenous SCF to cocultures prepared on Day 5 results in a transient but significant increase in the percentage of gonocytes with pseudopods even though we found that Sertoli cells in the cultures produce endogenous SCF. Thus, our findings provide evidence to support a role for c-kit expression by neonatal gonocytes and, presumably, SCF expression by neonatal Sertoli cells in stimulating migration of these germ cells in vitro.