Kate L. Loveland

Germ cell suicide: new insights into apoptosis during spermatogenesis

Mature sperm are the product of a precisely regulated developmental sequence in which germ cell proliferation, differentiation, self‐renewal and apoptosis are carefully controlled. The control of germ cell apoptosis during spermatogenesis is especially important. It is mediated by signals derived from the Sertoli cells with which each germ cell is closely associated, as well as by signals originating outside the testis. A greater understanding of these signals is emerging from studies of the spermatogenic defects of genetically modified animals. In particular, the intracellular signaling cascades which ultimately determine germ cell fate are being illuminated by recent studies of the Bcl‐2 protein family. This review summarises the crucial role which stringently regulated apoptosis plays in the production of male gametes. BioEssays 22:423—430, 2000.

Dicer1 Is Required for Differentiation of the Mouse Male Germline

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. Hundreds of miRNAs are expressed in mammals; however, their functions are just starting to be uncovered. MicroRNAs are processed from a long hairpin mRNA transcript, down to a ∼23-nucleotide duplex. The enzyme Dicer1 is required for miRNA processing, and mouse knockouts of Dicer1 are embryonic lethal before 7.5 days postcoitus. To examine the function of miRNAs specifically in the germline, we used a mouse model that expresses Cre recombinase from the TNAP locus and a floxed Dicer1 conditional allele. Removal of Dicer1 from germ cells resulted in male infertility. Germ cells were present in adult testes, but few tubules contained elongating spermatids. Germ cells that did differentiate to elongating spermatids exhibited abnormal morphology and motility. Rarely, sperm lacking Dicer1 could fertilize wild-type eggs to generate viable offspring. These results show that Dicer1 and miRNAs are essential for proper differentiation of the male germline.

The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis

BackgroundHistone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination.ResultsIn ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted.ConclusionThese data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

CHAPTER 18 – Cytology of the Testis and Intrinsic Control Mechanisms

The testis is surrounded by a dense connective tissue capsule, the tunica albuginea, which is covered on its anterior and lateral aspects with the remnants of the process vaginalis, forming the visceral and parietal layers of the tunica vaginalis. From the internal surface of the tunica albuginea, connective tissue septa extend posteriorly toward a region of the testis termed the mediastinum. This area consists of connective tissue within which an anastomotic network of ducts can be identified as the rete testis. The inner surface of the tunica albuginea is apposed to loose highly vascular connective tissue sometimes termed the tunica vasculosa. The degree of lobulation of the testis varies between species, and within these lobules lie the seminiferous tubules within which spermatogenesis occurs. The tubules extend as loops from the mediastinum testis, both ends of each loop communicating via single straight tubules, the tubuli recti. In most mammals, this simple arrangement is obscured in the adult testis, because the tubule forming each loop becomes extensively folded, thereby extending its surface area. The organization of the intertubular tissue also varies dramatically between species, but it contains the blood vessels, lymphatics, and nerve fibers. The Leydig cells are scattered in groups in the intertubular tissue in relation to the vasculature and the lamina propria of the seminiferous tubules, the outer layers of which consist of modified smooth muscle cells termed myoid cells.

Nuclear Transport of Parathyroid Hormone (PTH)-Related Protein Is Dependent on Microtubules

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.

Inhibin, activin, follistatin and FSH serum levels and testicular production are highly modulated during the first spermatogenic wave in mice.

Testicular development is governed by the combined influence of hormones and proteins, including FSH, inhibins, activins and follistatin (FST). This study documents the expression of these proteins and their corresponding mRNAs, in testes and serum from mice aged 0 through 91 days post partum (dpp), using real-time PCR, in situ hybridisation, immunohistochemistry, ELISA and RIA. Serum immunoactive total inhibin and FSH levels were negatively correlated during development, with FSH levels rising and inhibin levels falling. Activin A production changed significantly during development, with subunit mRNA and protein levels declining rapidly after 4 dpp, while simultaneously levels of the activin antagonists, FST and inhibin/activin beta(C), increased. Inhibin/activin beta(A) and beta(B) subunit mRNAs were detected in Sertoli, germ and Leydig cells throughout testis development, with the beta(A) subunit also detected in peritubular myoid cells. The alpha, beta(A), beta(B) and beta(C) subunit proteins were detected in Sertoli and Leydig cells of developing and adult mouse testes. While beta(A) and beta(B) subunit proteins were observed in spermatogonia and spermatocytes in immature testes, beta(C) was localised to leptotene and zygotene spermatocytes in immature and adult testes. Nuclear beta(A) subunit protein was observed in primary spermatocytes and nuclear beta(C) subunit in gonocytes and round spermatids. The changing spatial and temporal distributions of inhibins and activins indicate that their modulated synthesis and action are important during onset of murine spermatogenesis. This study provides a foundation for evaluation of these proteins in mice with disturbed testicular development, enabling their role in normal and perturbed spermatogenesis to be more fully understood.

Importin α mRNAS have distinct expression profiles during spermatogenesis

Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin α family genes. We first clarified the identity of the five known mouse importin α genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin α mRNAs were detected in spermatogonia. In the adult mouse testis, importins α1 and α3 were detected in spermatogonia and early pachytene spermatocytes. Importin α4 mRNA was identified in pachytene spermatocytes, α6 mRNA in round spermatids, and α2 mRNA in both of these. The distinct importin α expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis. Developmental Dynamics 235:253–262, 2006.

Concomitant loss of proapoptotic BH3-only Bcl-2 antagonists Bik and Bim arrests spermatogenesis

The BH3‐only proteins of the Bcl‐2 family initiate apoptosis through the activation of Bax‐like relatives. Loss of individual BH3‐only proteins can lead either to no phenotype, as in mice lacking Bik, or to marked cell excess, as in the hematopoietic compartment of animals lacking Bim. To investigate whether functional redundancy with Bim might obscure a significant role for Bik, we generated mice lacking both genes. The hematopoietic compartments of bik−/−bim−/− and bim−/− mice were indistinguishable. However, although testes develop normally in mice lacking either Bik or Bim, adult bik−/−bim−/− males were infertile, with reduced testicular cellularity and no spermatozoa. The testis of young bik−/−bim−/− males, like those lacking Bax, exhibited increased numbers of spermatogonia and spermatocytes, although loss of Bik plus Bim blocked spermatogenesis somewhat later than Bax deficiency. The initial excess of early germ cells suggests that spermatogenesis fails because supporting Sertoli cells are overwhelmed. Thus, Bik and Bim share, upstream of Bax, the role of eliminating supernumerary germ cells during the first wave of spermatogenesis, a process vital for normal testicular development.

Activin A Balances Sertoli and Germ Cell Proliferation in the Fetal Mouse Testis

Activin affects many aspects of cellular development, including those essential for reproductive fitness. This study examined the contribution of activin A to murine fetal testicular development, revealing contrasting outcomes of activin actions on Sertoli cells and gonocytes. Shortly after sex determination, from Embryonic Day 12.5 (E12.5) through to birth (0 dpp), the activin A subunit transcript (Inhba) level rises in testis but not ovary, followed closely by the Inha transcript (encoding the inhibitory inhibin alpha subunit). Activin receptor transcript levels also change, with Acvr1 (encoding ALK2) and Acvr2b (ActRIIB) significantly higher and lower, respectively, at 0 dpp compared with E13.5 and E15.5. Transcripts encoding the signaling mediators Smad1, Smad3, and Smad4 were higher at 0 dpp compared with E13.5 and E15.5, whereas Smad2, Smad5, and Smad7 were lower. Detection of phosphorylated (P-)SMAD2/3 in nearly all testis cell nuclei indicated widespread transforming growth factor beta (TGFB) and/or activin ligand signaling activity. In contrast to wild-type littermates, activin betaA subunit knockout (Inhba−/−) mice have significantly smaller testes at birth, attributable to a 50% lower Sertoli cell number and decreased Sertoli cell proliferation from E13.5. Inhba−/− testes contained twice the normal gonocyte number at birth, with some appearing to bypass quiescence. Persistence of widespread P-SMAD2/3 in Inhba−/− cells indicates other TGFB superfamily ligands are active in fetal testes. Significant differences in Smad and cell cycle regulator transcript levels correlating to Inhba gene dosage correspond to differences in Sertoli and germ cell numbers. In Inhba−/− testes, Cdkn1a (encoding p21cip1), identified previously in fetal gonocytes, was lower at E13.5, whereas Cdkn1b (encoding p27kip1 in somatic cells) was lower at birth, and cyclin D2 mRNA and protein were lower at E15.5 and 0 dpp. Thus, activin A dosage contributes to establishing the balance between Sertoli and germ cell number that is ultimately required for adult male fertility.

The role of activin, follistatin and inhibin in testicular physiology.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the beta(B) subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.