The connexins are a family of at least 15 proteins that form the intercellular membrane channels of gap junctions. Numerous connexins, including connexin43 (Cx43), have been implicated in reproductive processes by virtue of their expression in adult gonads. In the present study, we examined the gonads of fetal and neonatal mice homozygous for a null mutation in the Gja1 gene encoding Cx43 to determine whether the absence of this connexin has any consequences for gonadal development. We found that in both sexes at the time of birth, the gonads of homozygous mutants were unusually small. This appears to be caused, at least in part, by a deficiency of germ cells. The germ cell deficiency was traced back as far as Day 11.5 of gestation, implying that it arises during early stages of germ line development. We also used an organ culture technique to examine postnatal folliculogenesis in the mutant ovaries, an approach necessitated by the fact that Gja1 null mutant offspring die soon after birth because of a heart abnormality. The results demonstrated that folliculogenesis can proceed to the primary (unilaminar) follicle stage in the absence of Cx43 but that subsequent development is impaired. In neonatal ovaries of normal mice, Cx43 could be detected in the somatic cells as early as Day 1, when primordial follicles begin to appear, supporting the conclusion that this connexin is required for the earliest stages of folliculogenesis. These results imply that gap junctional coupling mediated by Cx43 channels plays indispensable roles in both germ line development and postnatal folliculogenesis.

No abstract

The advent of sexual reproduction and the evolution of a dedicated germline in multicellular organisms are critical landmarks in eukaryotic evolution. We report an ancient family of GCNA (germ cell nuclear antigen) proteins that arose in the earliest eukaryotes, and feature a rapidly evolving intrinsically disordered region (IDR). Phylogenetic analysis reveals that GCNA proteins emerged before the major eukaryotic lineages diverged; GCNA predates the origin of a dedicated germline by a billion years. Gcna gene expression is enriched in reproductive cells across eukarya -either just prior to or during meiosis in single-celled eukaryotes, and in stem cells and germ cells of diverse multicellular animals. Studies of Gcna-mutant C. elegans and mice indicate that GCNA has functioned in reproduction for at least 600 million years. Homology to IDRcontaining proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.

The temporal expression of type IV collagen, laminin, and entactin in the basal laminae of the seminiferous tubule during development of the mouse testis was determined. Northern blot analysis was used to examine changes in testicular mRNA for alpha 1-alpha 5 type IV collagen (IV) chains in mice ranging in age from newborn to adult (60 days). Levels for mRNA alpha 1(IV) and alpha 2(IV) chains were highest in newborns through Day 5 and remained elevated through Day 10, but then sharply declined to adult values by Day 30. In sharp contrast, alpha 3(IV) and alpha 4(IV) chain levels were low in newborns, peaked at Day 10, and then declined to adult values by Day 30. 5(IV) mRNA was elevated in newborns and at Days 5 and 10 before dropping to adult levels by Day 30. Changes in the deposition of alpha 1, alpha 2, and alpha 3(IV) collagen chains, laminin, and entactin into the inner and outer basal laminae of the seminiferous tubule were determined from the beginning of tubule formation (embryonic Day 12.5) through adulthood by immunofluorescence microscopy using polyclonal antibodies for these constituents. The alpha 1 and alpha 2(IV) chains, laminin, and entactin were deposited into the inner basal lamina at embyronic Day 12.5 and into the newly formed outer basal lamina at Day 5 after birth. The alpha 3(IV) chains were deposited into both the inner and outer basal laminae at Day 5. Thus, testicular alpha 1-alpha 3(IV) mRNA levels coincide with the incorporation of detectable collagen chains into the seminiferous basal laminae, suggesting transcriptional control of these alpha (IV) chains. Expression of of the alpha 3(IV) chain coincides with the initiation of spermatogenesis, suggesting a functional role of this chain in spermatogonial proliferation.

Northern blotting, in situ hybridization, and oligodeoxyribonucleotide excess solution hybridization were used to quantitate metallothionein-I (MT-I) and MT-II mRNAs in mouse testes. Testes from sexually mature adults contained high levels of both MT mRNAs (approximately 10-fold higher than those in control adult liver). Testicular MT mRNA levels were age dependent, being low the first 2 weeks after birth and increasing slowly thereafter to maximal levels in the adult (by 9 weeks after birth). In the adult testis, in situ hybridization indicated that only cells within the adluminal compartment (germ cells) of the seminiferous tubules contain high levels of MT mRNA. The appearance of cells containing elevated levels of MT mRNA during development was delayed from the onset of spermatogenesis. In situ hybridization suggested that MT mRNA accumulates after the initial differentiation of primary spermatocytes and is maintained in spermatids. Pachytene spermatocytes (PSC) and round spermatids (RTD) isolated from adult testes contained both MT-I and MT-II mRNAs in levels equivalent to those found in zinc-treated hepatocytes, whereas very low levels of MT mRNA were detected in isolated Sertoli cells (ST). In situ hybridization suggested that MT mRNA was present at only basal levels in interstitial, spermatogonial, and mature sperm cells at all developmental stages examined. Northern blot and in situ hybridization to sulfated glycoprotein-2 (SGP-2) mRNA, a ST-specific transcript, showed that SGP-2 mRNA is high in the testis of 1-week-old mice and decreases gradually to a lower level in the adult. In situ detection of this mRNA was consistent with the location of ST in the testis. SGP-2 mRNA was abundant in ST and rare in PSC and RTD preparations. Analysis of pulse-labeled proteins from isolated PSC and RTD indicated that these cells actively synthesize MT-I and MT-II. The high levels of MT mRNA in adult testes were not increased substantially after systemic injection of cadmium, zinc, or bacterial lipopolysaccharide. In marked contrast, these treatments led to dramatically increased levels of hepatic and ovarian MT mRNA. This study establishes that the MT genes are actively expressed in a developmentally regulated fashion in the male germ cells of the mouse. This suggests a role for MT in the process of spermatogenesis.

Seminiferous tubule basement membrane (STBM)plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six ␣-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of ␣3(IV) chain (40%) and the ␣4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six ␣(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of ␣(IV) chains, were characterized for their ␣(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with ␣(IV) chain-specific antibodies.

Mammalian primordial germ cells (PGCs) proliferate as they migrate from their initial location in the extraembryonic mesoderm to the genital ridge, the gonadal anlage. Once in the genital ridge, PGCs cease dividing and differentiate according to their gender. To identify ligands that might limit PGC proliferation, we analyzed growth factor receptors encoded in RNA obtained from purified germ cells shortly after their arrival in the genital ridge. Receptors for two members of the TGFbeta superfamily were found, TGFbeta1 and activin. As the signal-transducing domains of both receptor systems are highly conserved, the effects of both TGFbeta1 and activin on PGCs would be expected to be similar. We found that both ligands limited the accumulation of germ cells in primary PGC cultures. BrdU incorporation assays demonstrated that either ligand inhibits PGC proliferation. These results suggest that these signal transduction pathways are important elements of the mechanism that determines germ cell endowment.