A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes.

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 IDR-containing proteins implicated in DNA damage repair suggests that GCNA proteins may protect the genomic integrity of cells carrying a heritable genome.

A tissue retrieval and postharvest processing regimen for rodent reproductive tissues compatible with long-term storage on the international space station and postflight biospecimen sharing program.

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Long-term (6-wk) hindlimb suspension inhibits spermatogenesis in adult male rats.

The International Space Station will allow extended habitation in space and long-term exposure to microgravity (microG). A concern is the impact of long-term microG exposure on the ability of species to reproduce. The model often used to simulate microG is rat hindlimb suspension (HLS), where the hindlimbs are elevated above the cage floor with a tail harness. Experiments described here are the first to examine the effect of long-term HLS on testicular function in adult male rats. Free-roaming (controls), animals with only the tail harnessed but hindlimbs in contact with the cage floor (TO), and HLS animals were tested for 6 wk. Cryptorchidism was prevented in TO and HLS animals by partial constriction of the inguinal canal with sutures. All parameters were compared at the end of the 6-wk experiment. Testicular weights and spermatogenesis were significantly reduced by HLS, such that no spermatogenic cells beyond round spermatids were present and epididymides were devoid of mature sperm. In many tubules, loss of all germ cells, except a few spermatogonia, resulting in histopathology similar to the Sertoli cell, was observed. Spermatogenesis appeared unaffected in control and TO animals. Sertoli and Leydig cell appearance, testosterone, luteinizing hormone, and follicle-stimulating hormone levels, and epididymal and seminal vesicle weight were unchanged by HLS. Cortisone was not elevated by HLS; thus stress may not be a factor. These results demonstrate that spermatogenesis is severely inhibited by long-term HLS, whereas testicular androgen production is not. These results have significant implications regarding serious effects of long-term exposure to microG on the reproductive capability of scrotal mammals, including humans.

Connexin43 is expressed in mouse fetal ovary.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3betaHSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc. In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development.