Paula R. Brown

A-Kinase Anchoring Protein 4 Binding Proteins in the Fibrous Sheath of the Sperm Flagellum

Abstract The fibrous sheath is a unique cytoskeletal structure located in the principal piece of the sperm flagellum and is constructed of two longitudinal columns connected by closely spaced circumferential ribs. Cyclic AMP-dependent protein kinases are secured within specific cytoplasmic domains by A-kinase anchoring proteins (AKAPs), and the most abundant protein in the fibrous sheath is AKAP4. Several other fibrous sheath proteins have been identified, but how the fibrous sheath assembles is not understood. Yeast two-hybrid assays and deletion mutagenesis were used to identify AKAP4-binding proteins and to map the binding regions on AKAP4 and on the proteins identified. We found that AKAP4 binds AKAP3 and two novel spermatogenic cell-specific proteins, Fibrous Sheath Interacting Proteins 1 and 2 (FSIP1, FSIP2). Transcription of Akap4, Akap3, and Fsip1 begins in early spermatid development, whereas transcription of Fsip2 begins in late spermatocyte development. AKAP3 is synthesized in round spermatids and incorporated into the fibrous sheath concurrently with formation of the rib precursors. However, AKAP4 is synthesized and incorporated into the nascent fibrous sheath late in spermatid development. The AKAP4 precursor is processed in the flagellum and only the mature form of AKAP4 appears to bind AKAP3. These results suggest that AKAP3 is involved in organizing the basic structure of the fibrous sheath, whereas AKAP4 has a major role in completing fibrous sheath assembly.

Peritubular Myoid Cells Participate in Male Mouse Spermatogonial Stem Cell Maintenance

Peritubular myoid (PM) cells surround the seminiferous tubule and together with Sertoli cells form the cellular boundary of the spermatogonial stem cell (SSC) niche. However, it remains unclear what role PM cells have in determining the microenvironment in the niche required for maintenance of the ability of SSCs to undergo self-renewal and differentiation into spermatogonia. Mice with a targeted disruption of the androgen receptor gene (Ar) in PM cells experienced a progressive loss of spermatogonia, suggesting that PM cells require testosterone (T) action to produce factors influencing SSC maintenance in the niche. Other studies showed that glial cell line-derived neurotrophic factor (GDNF) is required for SSC self-renewal and differentiation of SSCs in vitro and in vivo. This led us to hypothesize that T-regulated GDNF expression by PM cells contributes to the maintenance of SSCs. This hypothesis was tested using an adult mouse PM cell primary culture system and germ cell transplantation. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when thymus cell antigen 1-positive spermatogonia isolated from neonatal mice were cocultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number and length of transplant-derived colonies was increased considerably by in vitro T treatment. These results support the novel hypothesis that T-dependent regulation of GDNF expression in PM cells has a significant influence on the microenvironment of the niche and SSC maintenance.

Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility

ABSTRACT Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4+/Gt). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4Gt/Gt) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4Gt/Gt mice compared to wild-type mice. Sperm from Eno4Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

Heat shock protein 2 promoter drives cre expression in spermatocytes of transgenic mice

We generated transgenic mouse line C57BL/6‐Tg(Hspa2‐cre)1Eddy/J (Hspa2‐cre), which expresses cre‐recombinase under the control of a 907‐bp fragment of the heat shock protein 2 (Hspa2) gene promoter. Transgene expression was determined using Gt(ROSA)26Sortm1Sor/J (ROSA26) and Tg(CAG‐Bgeo/GFP)21Lbe/J (Z/EG) reporter strains and RT‐PCR and immunohistochemistry assays. Hspa2‐cre expression mimicked the spermatogenic cell‐specific expression of endogenous HSPA2 within the testis, being first observed in leptotene/zygotene spermatocytes. Expression of the transgene also was detected at restricted sites in the brain, as occurs for endogenous HSPA2. Although the results of mating the Hspa2‐cre mice to mice with a floxed Cdc2a allele indicated that some expression of the transgene occurs during embryogenesis, the Hspa2‐cre mice provide a valuable new tool for assessing the roles of genes during and after meiotic prophase in pachytene spermatocytes. genesis 48:114–120, 2010. Published 2009 Wiley‐Liss, Inc.

Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice

The makings of the reproductive tract Every embryo, regardless of its sex, contains both male and female primitive reproductive tracts before sexual differentiation. To establish a sex-specific reproductive system, female embryos need to remove the components of male tracts. The general consensus contends that removal of the male tracts occurs by default, a passive outcome owing to a lack of testis-derived androgens. Working in mice, Zhao et al. discovered that this process instead was actively promoted by the transcription factor COUP-TFII (see the Perspective by Swain). Without the action of this factor, embryos retained male reproductive tracts, independently of androgen action. These findings unveil unexpected mechanisms underlying the sexually dimorphic establishment of reproductive tracts. Science, this issue p. 717; see also p. 648 A transcription factor drives the process by which female mouse embryos establish their sex-specific reproductive structures. The sexual differentiation paradigm contends that the female pattern of the reproductive system is established by default because the male reproductive tracts (Wolffian ducts) in the female degenerate owing to a lack of androgen. Here, we discovered that female mouse embryos lacking Coup-tfII (chicken ovalbumin upstream promoter transcription factor II) in the Wolffian duct mesenchyme became intersex—possessing both female and male reproductive tracts. Retention of Wolffian ducts was not caused by ectopic androgen production or action. Instead, enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium was responsible for the retention of male structures in an androgen-independent manner. We thus suggest that elimination of Wolffian ducts in female embryos is actively promoted by COUP-TFII, which suppresses a mesenchyme-epithelium cross-talk responsible for Wolffian duct maintenance.

Reporter Mice Express Green Fluorescent Protein at Initiation of Meiosis in Spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014.

Reproductive, Physiological, and Molecular Outcomes in Female Mice Deficient in Dhh and Ihh

Ovarian development requires coordinate communications among oocytes, granulosa cells, and theca cells. Two Hedgehog (Hh) pathway ligands, Desert hedgehog (Dhh) and Indian hedgehog (Ihh), are produced by the granulosa cells and work together to regulate theca cell specification and development. Mice lacking both Dhh and Ihh had loss of normal ovarian function, which raised the question of which biological actions are specifically controlled by each ligand during folliculogenesis. By comparing the reproductive fitness, hormonal profiles, and ovarian transcriptomes among control, Dhh single-knockout (KO), Ihh KO, and Dhh/Ihh double-knockout (DKO) mice, we examined the specific roles of Dhh and Ihh in these processes. Dhh/Ihh DKO female mice were infertile because of a lack of theca cells and their steroid product androgen. Although Dhh and Ihh KO mice were fertile with normal folliculogenesis, they had decreased androgen production and alterations in their ovarian transcriptomes. Absence of Ihh led to aberrant steroidogenesis and elevated inflammation responses, which were not found in Dhh KO mouse ovaries, implicating that IHH has a greater impact than DHH on the activation of the Hh signaling pathway in the ovary. Our findings provide insight into not only how the Hh pathway influences folliculogenesis but also the distinct and overlapping roles of Dhh and Ihh in supporting ovarian development.