Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.     

Is infertility a disease and does it matter?

Claims about whether or not infertility is a disease are sometimes invoked to defend or criticize the provision of state‐funded treatment for infertility. In this paper, I suggest that this strategy is problematic. By exploring infertility through key approaches to disease in the philosophy of medicine, I show that there are deep theoretical disagreements regarding what subtypes of infertility qualify as diseases. Given that infertility’s disease status remains unclear, one cannot uncontroversially justify or undermine its claim to medical treatment by claiming that it is or is not a disease. Instead of focusing on disease status, a preferable strategy to approach the debate about state‐funded treatment is to explicitly address the specific ethical considerations raised by infertility. I show how this alternative strategy can be supported by a recent theoretical framework in the philosophy of medicine which avoids the problems associated with the concepts of health and disease.     

Extra-pair copulations can insure female blue tits against male infertility

Mating outside the pair-bond occurs frequently in socially monogamous birds, but the benefits that females gain from this behaviour remain debated. One hypothesis is that females engage in extra-pair copulations (EPCs) to ensure that their clutch is fertilised in case their own mate is infertile, but evidence for this idea is scarce. We report on a case of an infertile male blue tit that bred in three successive years with three different females. In the first year, all eggs were sired by an extra-pair male whereas in the second year all eggs were unfertilised and contained no sperm. In the third year, the female produced two clutches that were fertilised by an extra-pair male, but – unlike ‘normal’ clutches – sperm numbers on the perivitelline membrane decreased rapidly over the laying sequence. Our findings show that blue tit females mated to an infertile male can escape reproductive failure by engaging in EPCs and support previous suggestions that EPCs in blue tits cease after the onset of egg laying.     

An intact Pms2 ATPase domain is not essential for male fertility

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2^ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2^ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2^cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2^cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2^cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2^ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2^cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3.     

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans

1. Download : Download high-res image (172KB)
2. Download : Download full-size image

     

Morphogenesis of the decapitated and decaudated sperm defect in two brothers

The “decapitated sperm” defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term “decapitated spermatozoa” is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as “decapitated tails” and “decaudated heads.” The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in the epididymis.     

Aneuploid spermatozoa in infertile men: teratozoospermia.

We and others have demonstrated that infertile men who are candidates for intracytoplasmic sperm injection (ICSI) have an increased frequency of chromosomal abnormalities in their sperm. Reports based on prenatal diagnosis of ICSI pregnancies have confirmed the increased frequency of chromosomal abnormalities in offspring. Most studies to date have lumped various types of infertility together. However, it is quite likely that some subsets of infertility have an increased risk of sperm chromosomal abnormalities whereas others do not. We have studied nine men with severe teratozoospermia (WHO, 1992 criteria, 0-13% morphologically normal forms) by multicolour fluorescence in situ hybridisation (FISH) analysis to determine if they have an increased frequency of disomy for chromosomes 13, 21, XX, YY, and XY, as well as diploidy. All of the men also had aesthenozoospermia (< 50% forward progression) but none of the men had oligozoospermia (<20 x 10(6) sperm/ml). The patients ranged in age from 20 to 49 years (mean 33.2 years) in comparison to 18 normal control donors who were 23 to 58 years (mean 35.6 years). The control donors had normal semen parameters and no history of infertility. A total of 180,566 sperm were scored in the teratozoospermic men with a minimum of 10,000 sperm analyzed/donor/chromosome probe. There was a significant increase in the frequency of disomy in teratozoospermic men compared to controls for chromosomes 13 (.23 vs.13%), XX (.13 vs.05%), and XY (.50 vs.30%) (P <.0001, 2-tailed Z statistic). This study indicates that men with teratozoospermia and aesthenozoospermia but with normal concentrations of sperm have a significantly increased frequency of sperm chromosomal abnormalities.