The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

Luteal cells are potent activators of T cell proliferation in vitro. The purpose of this study was to determine which subset of T cells is stimulated by luteal cells and whether luteal cell-induced T cell activation elicits a proinflammatory or anti-inflammatory T cell response. The first objective was to determine if luteal cell-stimulated T cell proliferation was mediated by class I or II major histocompatibility complex (MHC) molecules. T cell proliferation was inhibited by anti-MHC class I but not anti-MHC class II antibodies. The second objective was to determine which T cell subtype proliferates when cultured with luteal cells. The proportions of CD4 and CD8 cells were unchanged, but the number of gamma delta T cells was increased by coculture with luteal cells. Immunohistochemistry confirmed the presence of gamma delta T cells in midcycle and regressing corpus luteum. The final objective was to characterize T cell cytokine production stimulated by luteal cells. The concentrations of interferon-gamma (IFNG) and interleukin 10 (IL10) were increased in luteal cell-T cell cocultures, whereas IL4 was undetectable, and IL12 was barely detectable in culture medium. It was concluded that coculture of luteal cells and T cells resulted in activation of a somewhat unique T cell subset, gamma delta T cells, as well as production of both pro- and anti-inflammatory cytokines. To our knowledge, this is the first report of gamma delta T cell activation by luteal parenchymal cells of any species, raising the possibility that tissue-resident gamma delta T cells are involved in regulating the balance between tissue homeostasis and luteolysis.

Expression of procollagens (Col1a1/2, Col3a1, Col4a1/2, Col5a1/2) and fibronectin 1 (Fn1) in the mouse fetal placental tissue was examined during the second half of pregnancy. Ribonuclease protection assays (RPAs) revealed that levels of these mRNAs noticeably increased between Days 10 and 14 of pregnancy, and they remained at relatively constant levels thereafter. In situ hyridization showed that Col1a1 and Col4a1 mainly localized in the labyrinth, whereas Fn1 was expressed mainly in the spongiotrophoblast. Since members of the transforming growth factor-beta (TGFB) superfamily are involved in the regulation of extracellular matrix (ECM) expression in various tissues, mRNA levels of TGFB family members and their binding proteins were also examined by RPAs. Transforming growth factor-beta1–3 (Tgfb1–3), activin subunits (Inhba, Inhbb), follistatin (Fst), and follistatin-like 3 (Fstl3) were expressed in the placenta, whereas significant expression of myostatin (Mstn) was not detected. Although the expression patterns of Tgfb1–3 and Inhba in the placenta suggest possible involvement of TGFBs and activin A in the regulation of placental ECM expression, neither TGFBs nor activin A affected ECM mRNA levels in vitro. On the other hand, hypoxia significantly decreased Col1a1/2 and Col4a1/2 mRNAs in cultured placental cells, and a high-glucose condition significantly increased Col1a1 and Col3a1 mRNAs. Fn1 expression was increased under the high-glucose condition, although hypoxia also increased Fn1 expression to a lesser degree. These data suggest that an increase in oxygen tension and nutrient supply during placentation rather than TGFB family members may be responsible for the increase in the placental ECM mRNA expression.

Estradiol (E₂) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E₂-accelerated egg transport. Intrabursal application of OR486 prevented E₂ from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E₂ on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E₂ accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E₂ nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.

Sperm undergo extreme variations in temperature and osmolality during cryopreservation, resulting in cell damage that includes plasma membrane defects, changes in cell volume, decreased motility, and flagellar defects. However, the fundamental biologic mechanisms underlying these events are poorly understood. We investigated the effects of osmotic stress and cytochalasins b (CB) and d (CD), naturally occurring toxins that disrupt actin organization, on the actin cytoskeleton and motility of Rhesus macaque sperm (Macaca mulatta). Sperm were diluted in media of low, medium, or high osmolality, or medium-osmolality media containing CB or CD, were stained with phalloidin-fluorescein isothiocyanate, and were processed for microscopy. The majority of sperm incubated in medium-osmolality media exhibited postacrosomal stain, whereas the minority displayed banding patterns of F-actin stain in the head. High-osmolality media, as well as CB and CD incubation, resulted in reorganization of F-actin into bands of stain in the majority of sperm heads. Cytochalasin b treatment also resulted in curled and looped tails, a phenomenon of hyposmotic stress, and CB and CD caused significant, dose-dependent decreases in motility determined by computer-assisted sperm assessment. Rho A cell populations were determined using flow cytometry, and immunocytochemistry analysis demonstrated that Rho A localization was altered after osmotic stress. Together, our results support a mechanism in which reorganization of the actin cytoskeleton induced by osmotic stress and potentially mediated by a Rho A signaling pathway contributes to sublethal sperm flagellar and motility defects.

In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

Activation of rapid motility apparently is one of the first steps of sperm capacitation and can be studied in vitro. Previously we found that 2-chloro-2′-deoxyadenosine or the catecholamine isoproterenol activates mouse sperm motility in vitro via a pathway mediated by cAMP that requires extracellular Ca² , the atypical sperm adenylyl cyclase, and sperm-specific protein kinase A. We now show that several other adenosine analogs and catecholamines accelerate the flagellar beat of mouse and human sperm. Unexpectedly, the potent adenosine receptor agonist CGS21680 does not accelerate the beat, and the adenosine receptor antagonist DPCPX does not diminish the accelerating action of 2-chloro-2′-deoxyadenosine. The pharmacological profile for activation by catecholamines is also unusual. Both agonists and antagonists of beta-adrenergic receptors elevate the beat frequency. Moreover, both l-(−) and d-( ) isomers of epinephrine, norepinephrine, and isoproterenol produce similar acceleration of the beat. In contrast, inhibitors of equilibrative nucleoside transporters effectively slow the onset of the accelerating action of adenosine analogs. Replacement of external Na with Li also diminishes the accumulation of cAMP and slows the resultant accelerating action of 2-chloro-2′-deoxyadenosine, suggesting the involvement of a Na -dependent concentrative nucleoside transporter. Our results show that adenosine and catecholamine agonists act in a novel signaling pathway that does not involve G protein-coupled cell-surface receptors that link to conventional adenylyl cyclases. Instead, adenosine and analogs may be transported into sperm via equilibrative and concentrative nucleoside transporters to act on unknown intracellular targets.

Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1–3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1–3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.

Woodlands sheep have a putative genetic mutation (FecX2^W) that increases ovulation rate. At present, the identity of FecX2^W is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2^W could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2^W may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2^W ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2^W ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2^W ewes. FecX2^W ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2^W mutation.

To investigate the population-level effects of exposure to environmental endocrine disrupters, a mesocosm-scale study was carried out in which the reproductive performance of groups of free-spawning three-spined sticklebacks, Gasterosteus aculeatus, exposed as juveniles to a model estrogen, was assessed. Juvenile sticklebacks were exposed to ethinyl estradiol (EE₂) at measured concentrations of (mean ± SEM) 1.75 ± 0.37 ng L⁻¹ and 27.7 ± 1.08 ng L⁻¹ for 4 wk posthatch and then reared thereafter in pristine lake water until they reached adulthood. Exposure to the higher EE₂ concentration resulted in the occurrence of ovotestis among males, whereas no gonadal abnormalities were evident among males exposed to the lower concentration of EE₂. In addition, when spawning was allowed in the mesocosm environment, fewer nests were built per male, and fewer eggs were deposited per nest, in the group exposed to 27.7 ng L⁻¹. Males from this group also exhibited a less intense nuptial coloration than control males. In the group exposed to 1.75 ng L⁻¹ EE₂ posthatch, significantly fewer nests were built than in the control group. These results demonstrate that the timing of exposure to estrogenic contaminants, in developmental terms, is critically important. Short-term exposure to estrogens as juveniles can clearly influence reproductive performance as adults, despite all growth and development subsequent to the exposure period taking place in an estrogen-free environment. In addition, these results suggest that reproductive dysfunction can occur even in fish with no gross abnormalities in gonadal structure. This suggests that the absence of gonadal intersex is not a reliable indicator of the reproductive potential, or estrogen-exposure history, of fish populations or the only important factor involved in compromising the reproduction of estrogen-exposed fish.

Epigenetic reprogramming is thought to play an important role in the development of cloned embryos reconstructed by somatic cell nuclear transfer (SCNT). In the present study, dynamic reprogramming of histone acetylation and methylation modifications was investigated in the first cell cycle of cloned embryos. Our results demonstrated that part of somatic inherited lysine acetylation on core histones (H3K9, H3K14, H4K16) could be quickly deacetylated following SCNT, and reacetylation occurred following activation treatment. However, acetylation marks of the other lysine residues on core histones (H4K8, H4K12) persisted in the genome of cloned embryos with only mild deacetylation occurring in the process of SCNT and activation treatment. The somatic cloned embryos established histone acetylation modifications resembling those in normal embryos produced by intracytoplasmic sperm injection through these two different programs. Moreover, treatment of cloned embryos with a histone deacetylase inhibitor, Trichostatin A (TSA), improved the histone acetylation in a manner similar to that in normal embryos, and the improved histone acetylation in cloned embryos treated with TSA might contribute to improved development of TSA-treated clones. In contrast to the asymmetric histone H3K9 tri- and dimethylation present in the parental genomes of fertilized embryos, the tri- and dimethylations of H3K9 were gradually demethylated in the cloned embryos, and this histone H3K9 demethylation may be crucial for gene activation of cloned embryos. Together, our results indicate that dynamic reprogramming of histone acetylation and methylation modifications in cloned embryos is developmentally regulated.

CDB-4022, an indenopryridine, suppresses spermatogenesis and decreases inhibin secretion in adult male rats. In the present study, we investigated the effects of CDB-4022 on Leydig cell function. A single oral dose of CDB-4022 (2.5 mg/kg) resulted in a 2-fold decrease in serum testosterone levels after 7 days that was paralleled by a decrease in Cyp17a1 mRNA and protein levels and 17alpha hydroxylase enzymatic activity compared with vehicle-treated rats. Consistent with the lower serum testosterone levels, pituitary Lhb and Fshb mRNA levels were increased 3.2- and 2.3-fold, respectively, by CDB-4022 treatment. Ultrastructural analysis of pituitary gonadotrophs showed distended endoplasmic reticulum (ER) and fewer secretory granules in CDB-4022-treated rats, characteristic of enhanced secretory activity. Conversely, CDB-4022 increased serum progesterone levels, testicular Star mRNA and protein expression, and the number of Leydig cells per testis. Serum inhibin B levels were undetectable in CDB-4022-treated rats, while serum activin A levels were similar to controls, indicating that the CDB-4022-treated rats have an elevated activin A:inhibin B ratio. In the presence of hCG stimulation, activin A directly suppressed testosterone secretion but enhanced progesterone secretion from rat Leydig cell primary cultures. Likewise, treatment of MA-10 cells with activin A was found to enhance cAMP-stimulated progesterone secretion and STAR expression. Together, our data indicate that CDB-4022 treatment inhibits CYP17A1 and stimulates STAR expression, thereby decreasing testosterone but increasing progesterone production. We propose that unopposed actions of activin A most likely contribute to the steroid profile in rats after CDB-4022 treatment. Our findings establish CDB-4022 as a new model to examine intratesticular control mechanisms that modulate Leydig cell gene expression and function.

Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.

During spermatogenesis, spermatids undergo a “down and up” translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.

Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.

The vital importance of peroxisomal metabolism for regular function of the testis is stressed by the severe spermatogenesis defects induced by peroxisomal dysfunction. However, only sparse information is available on the role and enzyme composition of this organelle in distinct cell types of the testis. In the present study, we characterized the peroxisomal compartment in human and mouse testis in primary cultures of murine somatic cells (Sertoli, peritubular myoid, and Leydig cells) and in GFP-PTS1 transgenic mice with a variety of morphological and biochemical techniques. Formerly, peroxisomes were thought to be absent in late stages of spermatogenesis. However, our results obtained by detection of different peroxisomal marker proteins show the presence of these organelles in most cell types in the testis, except for mature spermatozoa. Furthermore, we demonstrate a strong heterogeneity of peroxisomal protein content in various cell types of the human and mouse testis and show marked differences in structure, abundance, and localization of these organelles in spermatids, depending on their maturation. Highest and selective enrichment of the peroxisomal lipid transporters (ABCD1 and ABCD3) as well as ACOX2, the key regulatory enzyme of the beta-oxidation pathway 2 for side chain oxidation of cholesterol, were found in Sertoli cells, whereas Leydig cells were enriched in catalase and ABCD2. Our results suggest a cell type-specific metabolic function of peroxisomes in the testis and point to an important role for peroxisomes in spermiogenesis and in the lipid metabolism of Sertoli cells.

Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GnRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GnRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotroph cells. Both cAMP and pituitary adenylyl cyclase-activating peptide increase GnRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GnRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GnRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotroph cells, alone and with GnRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of protein kinase A (PKA); GnRH1 stimulation was partially suppressed by either PKA or protein kinase C inhibitors. Only the proximal GnRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3′ NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GnRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GnRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GnRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.