The role of the mesonephros in cell differentiation and morphogenesis of the mouse fetal testis.

In mouse fetal gonads, the adjacent mesonephros is required for seminiferous cord formation in vitro (Buehr et al., Development 117: 273-281, 1993). Here, we have investigated the role of mesonephric cells in seminiferous cord formation and in differentiation of Sertoli and Leydig cells. Undifferentiated male gonads with and without mesonephros at 11.5 dpc were cultured and immunocytochemical staining of Müllerian-inhibiting substance (MIS) was used as the criterion for Sertoli cell differentiation. For Leydig cells, testosterone (T) radioimmunoassay and cytochemical detection of delta 5-3 beta-hydroxysteroid-dehidrogenase (HSD) were undertaken. An ultrastructural study was also performed. In our culture conditions, the timing of differentiation of both Sertoli and Leydig cells was similar to that in the fetus. Although mesonephros is required for seminiferous cord formation, differentiation of Sertoli and Leydig cells proceeds in its absence at 11.5 dpc. Moreover, when 3H-thymidine labeled mesonephroi were grafted to unlabeled gonads, endothelial and peritubular myoid-like cells migrated into the gonad. We propose that these cells might be the mesonephric cells required for seminiferous cord formation.

Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm.

In this study we have compared the in vitro differentiation potential of epiblast tissue from mouse and rat embryos. Epiblasts were isolated from egg cylinder stage embryos by microdissection and placed in culture. Rat cultures were distinguished by the copious production of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce parietal endoderm. This difference in capacity to regenerate extraembryonic endoderm marks a surprising distinction in development of the pluripotential lineage between these two closely related rodents.

An electrophoretically detectable modification of glucosephosphate isomerase in mouse spermatozoa.

Cellulose acetate electrophoresis of glucosephosphate isomerase (GPI) from mouse testis and spermatozoa revealed a minor band which is not seen in electropherograms of GPI from other tissues. The minor band is associated equally with the two electrophoretic variants of GPI examined. Treatment of tissue extracts with various enzymes, enzyme inhibitors or thiol reagents failed to alter the appearance or position of the minor bands in sperm and testis GPI, or to induce their appearance in GPI from other tissues. No minor bands were seen in electropherograms of testis GPI from immature males, or from males whose testes lack spermatozoa for genetic reasons.

Development of mouse germ cells in cultures of fetal gonads.

Mouse gonadal tissue was studied under various conditions of in vitro culture, with the aim of clarifying some of the somatic-cell influences that regulate the development of germ cells in the mammalian gonad. Gonadal ridges, with or without the adjacent mesonephric region, were removed from mouse embryos 10.5-12.5 days post coitum (dpc). In an organ culture system, the female ridges showed good development, with no masculinization. All germ cells entered meiosis at the expected time. Although some oocytes entered the growth phase, many primordial follicles were observed. 11.5- and 12.5-day male ridges formed testis cords, and the germ cells developed as T-prospermatogonia. In 10.5-day ridges, cells resembling Sertoli cells differentiated but did not form testis cords, and the germ cells entered meiosis. We conclude that full differentiation of the supporting cell lineage was not achieved when culture was begun at 10.5 dpc; our findings suggest that immature Sertoli cells neither form testis cords nor inhibit the entry of germ cells into meiosis. When the ridges were fragmented and cultured in gas-permeable dishes, the somatic cells grew out as a monolayer on which the germ cells rested. Under these conditions male germ cells did not enter meiosis and did not survive for more than a few days. Female germ cells entered meiosis. In contrast to the organ culture system, many of the surviving oocytes entered the growth phase during the second week of culture, reaching diameters of up to 60 microns. This suggests that normal follicular cell investment may play a crucial role in maintaining the oocyte in a state of developmental arrest. The growing oocytes showed the oocyte-specific expression of the enzyme glucose phosphate isomerase. It seems that the initiation and maintenance of both oocyte growth and oocyte-specific gene expression can take place in the absence of normal follicular cell investment.

Interlitter variation in progeny of chimaeric male mice.

Three chimaeric male mice (C57BL/6McL reversible BALB/c) were allowed to mate freely with BALB/c females, and produced two types of progeny: agouti (BALB/c egg X C57BL spermatozoon) and albino (BALB/c egg X BALB/c spermatozoon). The proportion of each type, however, varied significantly from litter to litter. The variation could not be related to the age of the male, or to the particular female who produced the litter, nor had it a clear cyclic pattern. Unilateral orchidectomy of one male resulted in a change in the overall frequency of progeny types observed, but did not alter the degree of interlitter variation. Females were then introduced to two of the males overnight (18:00-09:00 h) or during the day (09:00-18:00 h) in mouse rooms with a dark period from 19:00 to 05:00 h. Overnight matings produced a slight increase in the proportion of albino progeny, but daytime matings resulted in a significant increase in agouti young (P less than 0.001). The relative proportions of the two types of progeny in litters of these males therefore appeared to be affected by the time of mating relative to the time of ovulation in the female, suggesting that variation in the time of mating is responsible for the observed interlitter variation in progeny type. Electrophoretic analysis of GPI-1 from spermatogenic cells isolated from the testes of one of the chimaeric males after death showed that the proportions of the two cell types in the testes were closer to the progeny proportions obtained from overnight than from daytime matings.

Mesonephric contribution to testis differentiation in the fetal mouse.

Testes from 11.5-day-old mouse embryos, with and without attached mesonephroi, were cultured for 7 days. Isolated testes failed to develop well-differentiated testis cords: however, when cultured attached to a mesonephros from either a male or a female donor embryo, testes developed cords that were normal in appearance. Testes cultured next to a mesonephric region but separated from it by a permeable filter, did not develop normal cords, nor did testes grafted to fragments of embryonic limb or heart. When testes were grafted to mesonephric regions from mice carrying a transgenic marker, the marker was found in some of the peritubular myoid cells and other interstitial cells of the testis, but not in the Sertoli cells or the germ cells. We conclude that after 11.5 days post coitum, cells can migrate from the mesonephric region into the differentiating testis and can contribute to the interstitial cell population, and that this contribution is necessary for the establishment of normal cord structure. The germ cells in all cultured testes, whether or not differentiated cords were present, were T1 prospermatogonia: no meiotic germ cells were seen.

Proliferation and migration of primordial germ cells in We/We mouse embryos.

We have examined the numbers and distribution of primordial germ cells in We/We, We/+, and +/+ mouse embryos using Southern blotting to determine embryo genotypes. At early somite stages (5-7 somites: approximately 8 1/2 days post coitum [dpc]) there are 50 to 100 germ cells in embryos of all genotypes. The number of germ cells in We/+ and +/+ embryos then begins to increase: at later somite stages (17-19 somites: approximately 9 1/2 dpc) they number about 200, and by 10 1/2 dpc there are approximately 725 We/+ and 850 +/+ germ cells. During this time, however, the number of germ cells in We/We embryos remains less than 100. At 8 1/2 dpc, the distribution of germ cells in the hindgut endoderm is the same in all genotypes. By 9 1/2 dpc, 30% of We/We germ cells are found in ectopic sites (allantois and vitelline artery); germ cell distribution along the length of the hindgut appears normal, but germ cells remain confined to the floor of the gut in We/We embryos, rather than being distributed around its circumference as in the other two genotypes. By 10 1/2 days, the migration of We/We germ cells through the dorsal mesentery lags behind that of the other genotypes, and a larger proportion remains in the gut wall.

Low intracellular pH is involved in the early embryonic death of DDK mouse eggs fertilized by alien sperm.

Intracellular pH was measured in normal 8-cell stage mouse embryos and in embryos from a cross between DDK females and C3H males. DDK/C3H embryos display the DDK syndrome and spontaneously begin to decompact toward the late 16-cell stage. Ultimately, 90% fail to form blastocysts. Normal embryos have a resting intracellular pH close to neutrality. In DDK/C3H embryos a substantial proportion (46%) has an intracellular pH below 6.7. An equivalent proportion of DDK/C3H embryos was found previously to show slow communication through gap junctions at the 8-cell stage. This is probably a consequence of low intracellular pH. In normal embryos the weak acid, butyric acid, decreased intracellular pH and slowed the transfer of Lucifer Yellow through gap junctions. Normal embryos treated with butyrate for between 1 and 6 hr beginning at the 8-cell stage and cultured for 24 hr, reproduced the DDK/C3H phenotype. After 48 hr some butyrate treated embryos recovered, while others remained as decompacted morulae. Treatment of control and DDK/C3H 8-cell stage embryos with dibutyryl cyclic AMP or forskolin, which will increase intracellular cyclic AMP, speeded gap junctional communication. Forskolin treatment prevented expression of the DDK syndrome in DDK/C3H embryos, although the rescue was transient and the syndrome returned when forskolin was removed. The finding that the DDK syndrome is manifested as low intracellular pH may provide clues to the molecular basis of the defect.

Reduced gap junctional communication is associated with the lethal condition characteristic of DDK mouse eggs fertilized by foreign sperm.

Communication through gap junctions was examined in 8-cell zygotes generated by fertilization of eggs of the DDK inbred strain of mice with spermatozoa of the C3H strain. These zygotes spontaneously begin to extrude cells at the late 16-cell stage and 95% die by the blastocyst stage. The transfer of Lucifer Yellow between cells of DDK/C3H zygotes that had not yet begun to express the defect was significantly slower than in DDK/DDK controls or in controls from other strains. Treatment with the weak base methylamine, to raise intracellular pH, speeded the transfer of Lucifer in all strains; transfer between cells of DDK/C3H zygotes became as fast as that between cells of control zygotes. DDK/C3H zygotes cultured in methylamine either from the 4- to 8-cell stage to the early 16-cell stage (19h) or from the early to the late 16-cell stage (6 h) showed significant rescue to the blastocyst stage. Once spontaneous decompaction of cells from DDK/C3H zygotes had begun (the late 16-cell stage onwards) methylamine treatment was no longer able to bring about rescue. We conclude that zygotes developed from eggs of the DDK strain fertilized by foreign spermatozoa are characterized physiologically by defective gap junctional communication. Improving gap junctional communication is sufficient to allow many zygotes to maintain the compacted state, suggesting a link between compaction and communication through gap junctions.

XY follicle cells in ovaries of XX----XY female mouse chimaeras.

Oocytes with adhering follicle cells were sampled from ovaries obtained from 11 GPI-1A----GPI-1B chimaeras, comprising 10 females and 1 hermaphrodite. GPI analysis of individual oocytes revealed a marked bias towards the GPI-1B component in the germ line of this chimaeric combination. GPI-1B XY oocytes were identified in the ovary from the hermaphrodite, the bias towards the GPI-1B germ line perhaps helping to counterbalance the normally severe selection against XY oocytes. GPI analysis of follicle cells revealed a much more balanced contribution of the two components to this ovarian cell type. Importantly, GPI-1A follicle cells were identified in more than half the follicles from an XX----XY female in which the GPI-1A component was XY, supporting an earlier conclusion of Ford et al. (1974) that XY cells can contribute to the follicles of XX----XY female mice. It is suggested that XY cells can be recruited to form follicle cells in XX----XY chimaeras when there is a developmental mismatch between the two components, such that an ovary-determining signal produced by the XX component pre-empts the testis-determining action of the Y.

Immune reactivity of progeny of tetraparental male mice.

Steele and Gorczynski have recently suggested that inbred male mice rendered tolerant at birth to the alloantigens of an H-2 plus non-H-2 incompatible inbred strain can transmit this tolerance or hyporeactivity, as measured in a primary anti-H-2 cytotoxic T-cell test in vitro, to their progeny, born of normally reactive females syngeneic with the males. As a corollary, it might be expected that the progeny of tetraparental males which are tolerant because from earliest fetal development they are chimaeric with respect to all tissues, including haematopoietic cells and germ cells, might in turn be tolerant to the other set of paternal alloantigens. We have now found, on the contrary, that inbred progeny of one component of a tetraparental male showed heightened responsiveness to the other paternal alloantigens.

Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX----XY chimaeric mouse testes.

The distribution of XX and XY cells in XX----XY chimaeric mouse testes was analysed by enzyme marker analysis of separated testicular tissues and by in situ DNA marker analysis of air-dried testicular cells and testis sections. XX cells contributed to the Leydig cells, the peritubular cells and the vascularized connective tissue of the tunica albuginea. The Sertoli cells, on the other hand, appeared to be exclusively XY. These results indicate that during the development of the testis, Sertoli cell differentiation is triggered by cell-autonomous activity of the Y chromosomal testis-determining gene Tdy. Subsequent steps in testis differentiation may be a consequence of Sertoli cell activity.

Rapid loss of Oct-4 and pluripotency in cultured rodent blastocysts and derivative cell lines.

The POU transcription factor Oct-4 is essential for the pluripotent character of the mouse inner cell mass (ICM) and derivative embryonic stem (ES) cells. We analyzed the expression of Oct-4 during culture and establishment of cell lines from mouse and rat preimplantation embryos. Oct-4 was rapidly lost in primary outgrowths of the majority of cultured embryos prior to any evidence of morphological differentiation. Oct-4 persisted in only a minority of strain 129 cultures, which can go on to give ES cells. We used transgenic rats in which the dual reporter/selection marker beta-geo is under control of Oct-4 regulatory elements to investigate the effect of direct selection for Oct-4 expressing cells. Ablation of all cells occurred, consistent with complete downregulation of Oct-4. Without selection, in contrast, continuous cultures of morphologically undifferentiated cells could be derived readily from rat blastocysts and ICMs. However, these cells did not express significant Oct-4 and, although capable of differentiating into extraembryonic cell types, appeared incapable of producing fetal germ layer derivatives. Downregulation of Oct-4 appears to be a limiting factor in attempts to derive pluripotent cell lines from preimplantation embryos.