Sperm GIRK2-containing K+ inward rectifying channels participate in sperm capacitation and fertilization.

The GIRK2-containing inward-rectifying K(+) ion channels have been implicated in mammalian spermatogenesis. While the Girk2 null mice are fertile, the male weaver transgenic mice carrying a gain-of-function mutation in the Girk2 gene are infertile. To establish the exact period of spermatogenesis affected by this mutation, we performed StaPut isolation and morphological characterization of the germ cells present in the weaver testis. Germ cells representing all periods of spermatogenesis were identified. However, no spermatozoa were present, suggesting that this mutation only affected the haploid phase of spermatogenesis. Real-time PCR studies performed on StaPut purified germ cells from wild-type mice indicated that the Girk2 transcripts were exclusively expressed in spermatids. Immunofluorescence studies of mouse and boar spermatids/spermatozoa localized the GIRK2 K(+) containing channels to the acrosomal region of the sperm plasma membrane. During porcine in vitro fertilization (IVF), GIRK2-containing channels remained associated with the acrosomal shroud following zona-induced acrosome reaction. Fertilization was blocked by tertiapin-Q (TQ), a specific inhibitor of GIRK channels, and by anti-GIRK2 antibodies. Altogether, studies in two different mammalian species point to a conserved mechanism by which the GIRK2 inward-rectifying K(+) ion channels support sperm function during fertilization.

Developmental patterns of PPAR and RXR gene expression during spermatogenesis.

Members of the family of nuclear receptors that include peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are important mediators of selective gene activation during development and cellular differentiation. In this study, developmentally-specific PPAR and RXR patterns of expression that occur in somatic and germ cell populations in the testis were determined using quantative real-time PCR (qRT-PCR) studies on RNAs that were isolated from StaPut purified mouse germ cells and primary rat Sertoli cells. These qRT-PCR studies indicate that transcripts encoding the PPAR-Alpha (α), -Beta (ß), and -Gamma (γ) and RXR -Alpha (α), -Beta (ß), and -Gamma (γ) are developmentally expressed in both differentiating germ and Sertoli cells. In further experiments aimed at deciphering the physiological role that PPAR-Gamma (γ) plays in Sertoli cells, 15-day primary rat Sertoli cells were infected with recombinant adenoviral vectors containing PPAR-Gamma (γ) cDNA and PPAR-Gamma (γ) RNAi constructs. Affymetrix microarray analysis and qRT-PCR validation studies using total RNA isolated from these transfected cells indicated that PPAR-Gamma regulates the pattern of expression of key lipid metabolic genes in Sertoli cells.

Molecular and functional characterization of the murine ldh2 promoter region: Sp-binding GC-box domains are the key cis-elements regulating ldh2 gene expression during spermatogenesis.

The goal of the present study was to elucidate the specific transcriptional mechanisms that regulate ldh2 gene expression during the early stages of spermatogenesis. DNA sequence analysis of the 1.0-kb ldh2 promoter region directly upstream of the transcriptional start site indicated the presence of three SP-protein binding GC-box elements and the absence of TATA and CAAT boxes. Functional characterization studies of the mouse ldh2 promoter were performed in the SV40 transformed mouse spermatogonial cell line, GC-1 spg. Transfection/transient expression studies using full-length and truncated ldh2 promoter/luciferase reporter constructs revealed that all three of the SP-binding cis-regulatory GC-box elements are required for optimal ldh2 promoter activity. Additional site-directed mutagenesis studies indicated that the two most proximal GC-box sites play essential regulatory roles in mediating basal ldh2 promoter activity. These studies suggest that the expression of the ldh2 gene in spermatogonia and early spermatocytes are regulated by SP-mediated transcriptional mechanisms.

SP1 transcription factors in male germ cell development and differentiation.

Transcription factor SP1 is a zinc finger protein that has been implicated in regulating the expression of several genes involved in cellular differentiation and embryonic development. The zinc finger region of SP1 transcription factors binds to GC or GT-box elements present in the promoters of a number of male germ cell target genes that are developmentally expressed during spermatogenesis. The glutamine and serine/threonine-rich regions of the SP1 proteins recruit co-regulatory factors to the multi-protein preinitiation complex that are important for mediating transcriptional activation in male germ cells. Studies in our laboratory have identified several alternatively spliced transcripts encoding SP1 isoforms that display stage and cell-type-specific expression profiles in differentiating germ cells in the seminiferous epithelium of the testis. This review summarizes the expression patterns and functional significance of these SP1 transcription factor variants during spermatogenesis.

Identification, characterization, and functional analysis of sp1 transcript variants expressed in germ cells during mouse spermatogenesis.

The SP family of zinc-finger transcription factors are important mediators of selective gene activation during embryonic development and cellular differentiation. SP-binding GC-box domains are common cis-regulatory elements present in the promoters of several genes expressed in a developmentally specific manner in differentiating mouse germ cells. Four Sp1 cDNAs were isolated from a mouse pachytene spermatocyte cDNA library and characterized by DNA sequence analysis. Northern blot studies revealed that these cDNAs corresponded to 3 full-length Sp1 transcripts (4.1, 3.7, and 3.2 kilobases [kb]) and an additional 1.4-kb 5'-truncated Sp1 transcript that are temporally expressed during spermatogenesis. Quantitative real-time polymerase chain reaction studies verified that the highest levels of Sp1 transcript expression of 4.1, 3.7, and 3.2 kb occur in the primary spermatocytes. The spatial and temporal expression patterns of these Sp1 transcripts and their encoded 60-kDa and 90-kDa SP1 proteins were demonstrated using in situ hybridization and immunohistochemical analyses. To assess the transcriptional properties of these SP1 transcription factors, SP-deficient Drosophila SL2 cells were stably transfected with the respective Sp1 cDNA expression vectors and cotransfected with either Ldh2, Ldh3, or Creb promoter/luciferase reporter constructs. The levels of SP-mediated luciferase expression observed depended on the structure of the glutamine-rich transactivation domains and the number of GC-box elements present in the respective promoters. The alterations observed in germ cells in the patterns of expression of the Sp1 transcripts encoding the 60-kDa and 90-kDa SP1 isoforms suggest that these SP1 factors may be involved in mediating stage-specific and cell type-specific gene expression during mouse spermatogenesis.