Drug transporters, the blood-testis barrier, and spermatogenesis

The blood-testis barrier (BTB), which is created by adjacent Sertoli cells near the basement membrane, serves as a 'gatekeeper' to prohibit harmful substances from reaching developing germ cells, most notably postmeiotic spermatids. The BTB also divides the seminiferous epithelium into the basal and adluminal (apical) compartment so that postmeiotic spermatid development, namely spermiogenesis, can take place in a specialized microenvironment in the apical compartment behind the BTB. The BTB also contributes, at least in part, to the immune privilege status of the testis, so that anti-sperm antibodies are not developed against antigens that are expressed transiently during spermatogenesis. Recent studies have shown that numerous drug transporters are expressed by Sertoli cells. However, many of these same drug transporters are also expressed by spermatogonia, spermatocytes, round spermatids, elongating spermatids, and elongated spermatids, suggesting that the developing germ cells are also able to selectively pump drugs 'in' and/or 'out' via influx or efflux pumps. We review herein the latest developments regarding the role of drug transporters in spermatogenesis. We also propose a model utilized by the testis to protect germ cell development from 'harmful' environmental toxicants and xenobiotics and/or from 'therapeutic' substances (e.g. anticancer drugs). We also discuss how drug transporters that are supposed to protect spermatogenesis can work against the testis in some instances. For example, when drugs (e.g. male contraceptives) that can perturb germ cell adhesion and/or maturation are actively pumped out of the testis or are prevented from entering the apical compartment, such as by efflux pumps.     

Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro

Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm. Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spermatogenesis in vitro and to develop approaches for germ-line transmission.     

Blanks, a nuclear siRNA/dsRNA-binding complex component, is required for Drosophila spermiogenesis

Small RNAs and a diverse array of protein partners control gene expression in eukaryotes through a variety of mechanisms. By combining siRNA affinity chromatography and mass spectrometry, we have identified the double-stranded RNA-binding domain protein Blanks to be an siRNA- and dsRNA-binding protein from Drosophila S2 cells. We find that Blanks is a nuclear factor that contributes to the efficiency of RNAi. Biochemical fractionation of a Blanks-containing complex shows that the Blanks complex is unlike previously described RNA-induced silencing complexes and associates with the DEAD-box helicase RM62, a protein previously implicated in RNA silencing. In flies, Blanks is highly expressed in testes tissues and is necessary for postmeiotic spermiogenesis, but loss of Blanks is not accompanied by detectable transposon derepression. Instead, genes related to innate immunity pathways are up-regulated in blanks mutant testes. These results reveal Blanks to be a unique component of a nuclear siRNA/dsRNA-binding complex that contributes to essential RNA silencing-related pathways in the male germ line.     

In-vitro maturation of immature human male germ cells

Both meiotic and postmeiotic maturation events have been observed to occur in human male germ cells during in-vitro culture. The temperature of 30 degrees C, medium supplementation with follicle-stimulating hormone and testosterone and the maintenance of the original cell-cell associations within explanted segments of the testicular seminiferous tubules are common features of the most efficient culture systems. The in-vitro maturation processes are markedly accelerated as compared to the in-vivo situation, probably due to the abrogation of a checkpoint controlling the full assembly of molecules needed for spermiogenesis. Moreover, both meiotic and postmeiotic maturation processes can be reactivated in vitro in some cases with a complete block of the same processes in vivo. Healthy babies were born after micromanipulation-assisted fertilization with in-vitro matured elongated spermatids from men with complete in-vivo maturation arrest at the round spermatid stage and at the primary spermatocyte stage.     

Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice

We identified a testis-specific gene encoding a protein containing a BTB/POZ domain and six kelch repeats, which we named kelch homolog 10 (KLHL10). KLHL10 displays high evolutionary conservation in mammals, as evidenced by 98.7% amino acid identity between mouse and human KLHL10. KLHL10 is exclusively expressed in the cytoplasm of elongating and elongated spermatids (steps 9-16). We generated a Klhl10 null allele in 129S6/SvEv mouse embryonic stem cells, and obtained 47 chimeras from six independent embryonic stem cell lines. Whereas low-percentage male chimeras only produce C57BL/6J offspring, high-percentage chimeric and heterozygous males were completely infertile because of disrupted spermiogenesis characterized by asynchronous spermatid maturation, degeneration of late spermatids, sloughing of postmeiotic germ cells from the seminiferous epithelium, and marked reduction in the numbers of late spermatids. Our data demonstrate that, like protamine-1 and -2, both alleles of Klhl10 are required for male fertility and that haploinsufficiency caused by a mutation in one allele of Klhl10 prevents genetic transmission of both mutant and WT alleles.     

Infertility and testicular defects in hormone-sensitive lipase-deficient mice

The 84-kDa hormone-sensitive lipase (gene designation Lipe; EC 3.1.1.3) is a cholesterol esterase and triglyceride hydrolase that functions in the release of fatty acids from adipocytes. The role of hormone-sensitive lipase in other tissues such as the testis, where a specific 120-kDa testis-specific isoform is expressed, is unknown. To study this, we examined the fertility and testicular histology of gene-targeted hormone-sensitive lipase-deficient mice. Homozygous hormone-sensitive lipase-deficient male mice are infertile and have decreased testis weights; female homozygotes are fertile. Testicular abnormalities, detected at the light and electron microscopic levels, included the presence of multinucleated round and elongating spermatids, vacuolization of the seminiferous epithelium, asynchronization of the spermatogenic cycle, sloughing of postmeiotic germ cells from the seminiferous epithelium into the lumen, and a marked reduction in the numbers of late spermatids. Extensive nuclear head deformation was noted in late spermatids as well as the sharing of a common acrosome in multinucleated cells. In some multinucleated cells, nuclei were separated from their acrosomes, with the acrosomes remaining attached to areas of ectoplasmic specializations, suggesting defects in intercellular cytoplasmic bridge integrity. Although the lumen of the epididymis was essentially devoid of spermatozoa and filled instead with spherical degenerating cells, the epididymal epithelial cells appeared normal. The few late spermatids present in the epididymis were abnormal. There was no morphological evidence, as judged by the absence of lipid droplets of triacylglycerol or cholesteryl ester accumulation in the testis. Together, the data suggest that hormone-sensitive lipase deficiency results in abnormalities in spermiogenesis that are incompatible with normal fertility. We speculate that a metabolite downstream from the hormone-sensitive lipase reaction may be essential for membrane stabilization and integrity in the seminiferous epithelium and, in particular, may play an important role in the maintenance of intercellular cytoplasmic bridges between postmeiotic germ cells.     

The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis

Spermatogenesis is thought to critically depend on the high intratesticular testosterone (T) levels induced by gonadotropic hormones. Strategies for hormonal male contraception are based on disruption of this regulatory mechanism through blockage of gonadotropin secretion. Although exogenous T or T plus progestin treatments efficiently block gonadotropin secretion and suppress testicular T production, only approximately 60% of treated Caucasian men reach contraceptive azoospermia. We now report that in luteinizing hormone receptor knockout mice, qualitatively full spermatogenesis, up to elongated spermatids of late stages 13-16, is achieved at the age of 12 months, despite absent luteinizing hormone action and very low intratesticular T (2% of control level). However, postmeiotic spermiogenesis was blocked by the antiandrogen flutamide, indicating a crucial role of the residual low testicular T level in this process. The persistent follicle-stimulating hormone action in luteinizing hormone receptor knockout mice apparently stimulates spermatogenesis up to postmeiotic round spermatids, as observed in gonadotropin-deficient rodent models on follicle-stimulating hormone supplementation. The finding that spermatogenesis is possible without a luteinizing hormone-stimulated high level of intratesticular T contradicts the current dogma. Extrapolated to humans, it may indicate that only total abolition of testicular androgen action will result in consistent azoospermia, which is necessary for effective male contraception.     

Birth of normal young after electrofusion of mouse oocytes with round spermatids.

Normally, round spermatids, the youngest male germ cells with a set of haploid chromosomes, cannot fertilize mature oocytes. However, when mouse spermatids were fused with oocytes, some of the resulting zygotes developed into normal fertile mice of either sex. This demonstrates that the nuclei of spermatids can provide the paternally imprinted chromosomes needed for full embryonic development and also that the complex postmeiotic modifications involved in sperm formation in the testis and their maturation in the epididymis merely serve to facilitate natural delivery of the paternal genome. This finding may find an application in the treatment of male infertility due to defective spermiogenesis/sperm maturation.     

Expression of c-mos RNA in germ cells of male and female mice.

We have investigated the cell types in mouse testis and ovary in which the c-mos protooncogene is normally transcribed. Blot hybridization analysis of electrophoretically fractionated RNAs from testes of mice with defects in germ-cell development and from prepubertal and adult mice indicated that c-mos was transcribed during male germ-cell development. Analysis of purified populations of spermatogenic cell types detected c-mos RNA in the earliest haploid postmeiotic germ cell, the round spermatid, indicating that c-mos was expressed transiently during spermatogenesis. c-mos RNA was detected by blot hybridization in the ovaries of prepubertal mice and decreased in relative concentration following gonadotropin-stimulated proliferation of granulosa cells. These results suggested that c-mos was transcribed in oocytes and were confirmed by detection of high levels of c-mos RNA in isolated grown oocytes. Thus, c-mos is expressed in both male and female germ cells, suggesting possible roles for this protooncogene in meiosis, germ-cell development, fertilization, and early embryogenesis.