A heteromeric integral membrane protein, Na /K ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na /K ATPase, to Na /K ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na /K ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na /K ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na /K ATPase. Binding of ouabain to Na /K ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na /K ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na /K ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na /K ATPase in mammalian sperm capacitation.

Spermatogenic cells elaborate a highly specialized differentiation program that is mediated in part by germ cell-enriched transcription factors. This includes a novel member of the sterol response element-binding factor family, SREBF2_v1/SREBP2gc. Somatic SREBFs are predominantly synthesized as precursor proteins and are critical regulators of cholesterol and fatty acid synthesis. In contrast, SREBF2_v1 bypasses the precursor pathway and has been directly implicated in spermatogenic cell-specific gene expression. During spermatogenesis, SREBF2 precursor transcripts predominate in premeiotic stages, while SREBF2_v1 is highly upregulated specifically in pachytene spermatocytes and round spermatids. In the present study, we demonstrate that Srebf2_v1 mRNAs are present in the testis of several mammalian species, including humans. The basis for the stage-dependent transition in SREBF2 isoforms was also investigated. A 3' rapid amplification of cDNA ends (RACE)-PCR analysis of the rat and human revealed that Srebf2_v1 transcripts are generated by alternative pre-mRNA cleavage/polyadenylation. This involves the use of an intronic, A(A/U)UAAA-independent poly(A) signal within intron 7 of the Srebf2 gene. Developmentally regulated competition between germ cell factors that control RNA splicing and pre-mRNA cleavage/polyadenylation may underlie this process. These results define an important role for alternative polyadenylation in male germ cell gene expression and development by controlling a stage-dependent switch in transcription factor structure and function during spermatogenesis. The Srebf2 gene thus provides a useful model to explore the role of alternative polyadenylation in regulating stage-dependent functions of important protein regulators in spermatogenic cells.

The activating protein 2 (AP-2) transcription factor family is required for multiple aspects of mouse postimplantation development, but much less is known about the expression and possible function of these genes during the preimplantation period. In the present study, we have examined the expression of all five members of the mouse AP-2 gene family in the unfertilized oocyte and from zygote formation to the blastocyst stage of development. Four AP-2 genes are differentially expressed during the preimplantation period, Tcfap2a, Tcfap2b, Tcfap2c, and Tcfap2e. Furthermore, with the exception of Tcfap2a, these genes are also expressed in unfertilized oocytes, indicating that they may be important for oogenesis, maternal-effect functions, or both. Given these findings, we have initiated studies to assess how various combinations of maternal and zygotic AP-2 gene expression might function together to regulate pre- and peri-implantation development. The present study focuses on the interplay between the expression of zygotic Tcfap2a and maternal and zygotic Tcfap2c. These studies indicate that zygotic, but not maternal, Tcfap2c expression is required for normal embryogenesis. In addition, the combined loss of both Tcfap2a and Tcfap2c accelerates embryonic lethality compared to the loss of either gene alone, demonstrating that genetic redundancy exists between these two AP-2 family members during the peri-implantation period of embryogenesis.

Regulation of the contractile effects of tachykinins and histamine on the human uterus was investigated with biopsy sections of the outer myometrial layer. The effects of neurokinin A (NKA) and human hemokinin-1 (hHK-1) in tissues from pregnant but not from nonpregnant women were enhanced by the inhibition of neprilysin. The effects of NKA and eledoisin were blocked by the NK₂ receptor antagonist SR 48968 but not by the NK₁ receptor antagonist SR 140333 in tissues from both groups of women. Human HK-1 acted as a partial agonist blocked by SR 48968 and, to a lesser extent, by SR 140333; endokinin D was inactive. In tissues from pregnant women, responses to high potassium-containing Krebs solution were 2–3-fold higher than those from nonpregnant women. Mepyramine-sensitive maximal responses to histamine were similarly enhanced. The absolute maximum responses to NKA and its stable NK₂ receptor-selective analogue, [Lys⁵MeLeu⁹Nle¹⁰]NKA(4–10), were increased in pregnancy, but their efficacies relative to potassium responses were decreased. Tachykinin potencies were lower in tissues from pregnant women than in those from nonpregnant women. These data 1) show for the first time that hHK-1 is a uterine stimulant in the human, 2) confirm that the NK₂ receptor is predominant in mediating tachykinin actions on the human myometrium, and 3) indicate that mammalian tachykinin effects are tightly regulated during pregnancy in a manner that would negate an inappropriate uterotonic effect. The potencies of these peptides in tissues from nonpregnant women undergoing hysterectomy are consistent with their possible role in menstrual and menopausal disorders.

Metformin is an anti-diabetic drug commonly used to treat cycle disorders and anovulation in women with polycystic ovary syndrome. However, the effects and molecular mechanism of metformin in the ovary are not entirely understood. We investigated the effects of this drug on steroidogenesis and proliferation in rat granulosa cells. Metformin (10 mM) treatment for 48 h reduced progesterone and estradiol (E₂) production in both basal conditions and under FSH stimulation. It also decreased the levels of the HSD3B, CYP11A1, STAR, and CYP19A1 proteins in response to FSH (10⁻⁸ M) and of HSD3B in the basal state only. Metformin treatment (10 mM, 24 h) also reduced cell proliferation and the levels of CCND2 and CCNE proteins without affecting cell viability, both in the basal state and in response to FSH. Furthermore, metformin treatment for 1 h simultaneously increased the Thr172 phosphorylation of PRKAA (adenosine 5′ monophosphate-activated protein kinase alpha) and the Ser79 phosphorylation of ACACA (acetyl-Coenzyme A carboxylase alpha). The adenovirus-mediated production of dominant-negative PRKAA totally abolished the effects of metformin on progesterone secretion, HSD3B and STAR protein production, and MAPK3/1 phosphorylation. Conversely, total inhibition of PRKAA Thr172 phosphorylation with the dominant-negative PRKAA adenovirus did not restore the decrease in E₂ production and cell proliferation induced by metformin. Our results therefore strongly suggest that metformin reduces progesterone production via a PRKAA-dependent mechanism, whereas PRKAA activation is not essential for the decrease in E₂ production and cell growth induced by metformin in rat granulosa cells.

In Sertoli cells of testis, androgen receptor-regulated gene transcription plays an indispensable role in maintaining spermatogenesis. Androgen receptor activity is modulated by a number of coregulators which are associated with the androgen receptor. Non-POU-domain-containing, octamer binding protein (NONO), a member of the DBHS-containing proteins, complexes with androgen receptor and functions as a coactivator for the receptor. Paraspeckle protein 1 alpha isoform (PSPC1, previously known as PSP1) and Splicing factor, proline- and glutamine-rich (SFPQ, previously known as PSF), other members of the DBHS-containing proteins, are also found in androgen receptor complexes, suggesting that these DBHS-containing proteins may cooperatively regulate androgen receptor-mediated gene transcription. We demonstrated that PSPC1, NONO, and SFPQ are coexpressed in Sertoli cell line TTE3 and interact reciprocally. The effect of the DBHS-containing proteins on the transcriptional activity was assessed using the construct containing androgen-responsive elements followed by a luciferase gene. The results showed that all the DBHS-containing proteins activate androgen receptor-mediated transcription, and PSPC1 is the most effective coactivator among them. Furthermore, we confirmed the presence of PSPC1, NONO, and SFPQ proteins in Sertoli cells of adult mouse testis sections. These observations suggest that PSPC1, NONO, and SFPQ form complexes with each other in Sertoli cells and may regulate androgen receptor-mediated transcriptional activity.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most of the toxic and endocrine-disruptive actions of aromatic compounds in the ovary. Paradoxically, this receptor has been shown to play important roles in normal female reproductive function as well. Although knowledge of AHR expression regulation in the ovary is of crucial significance to understand the receptor biology and its function in reproductive physiology, there are only limited data in this area. The purpose of the present study was to establish the possible regulation that AHR might undergo in ovarian cells. Here we show that the hormones FSH and estradiol are able to reduce AHR protein and transcript levels in granulosa cells in a way that parallels the changes observed in ovarian tissue across the rat estrous cycle. These findings suggest that estradiol and FSH would be cycle-associated endogenous modulators of AHR expression. In addition, we show that in granulosa cells the receptor is rapidly downregulated via proteasomal degradation following treatment with AHR ligands. However, prolonged treatment with an agonist caused an increase in Ahr mRNA levels. These actions would constitute a regulatory mechanism that both attenuates AHR signal rapidly and replenishes the cellular receptor pool in the long term. In conclusion, our results indicate that AHR expression is regulated by classical hormones and by its own ligands in granulosa cells.

The prostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.

In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 × 10⁷ cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 × 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.

We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The K_m for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6¾ pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6¾ pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.

Neurotransmitter-controlled Cl⁻ secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca² activated Cl⁻ channel blocker disulfonic acid stilbene (DIDS, 300 μM) only inhibited part of the CCH-induced Isc response, while nonselective Cl⁻ channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 μM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca² -chelating agent BAPTA/AM (50 μM) and an endoplasmic reticulum Ca² pump inhibitor, Thapsigagin (Tg, 1 μM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl⁻ secretion could be mediated by Ca² and cAMP-dependent regulatory pathways.

Early pregnancy loss is the most common complication of human reproduction. Given the complexities of early development, it is likely that many mechanisms are involved. Knowledge of differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying early pregnancy loss. To identify proteins with different expression profiles related to early pregnancy loss, we applied a proteomic approach and performed two-dimensional gel electrophoresis (2-DE) on six placental villous tissues from patients with early pregnancy loss and six from normal pregnant women, followed by comparison of the silver-stained 2-DE profiles. It was found that 13 proteins were downregulated and 5 proteins were upregulated significantly (P < 0.05) in early pregnancy loss as determined by spot volume. Among them, 10 downregulated and 2 upregulated spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anomalies of these proteins, including three principal antioxidant enzymes (copper/zinc-superoxide dismutase, peroxiredoxin 3, and thioredoxin-like 1 protein), S100 calcium binding protein, galectin-1, chorionic somatomammotropin hormone 1, transthyretin, fas inhibitory molecule, eukaryotic translation elongation factor, RNA-binding protein, ubiquitin-conjuating enzyme E2N, and proteasome beta-subunit, indicate widespread failure in cell regulations and processes such as antioxidative defense, differentiation, cell proliferation, metabolism, apoptosis, transcription, and proteolysis in early pregnancy loss. This study has identified several proteins that are associated with placentation and early development, shedding a new insight into the proteins that may be potentially involved in the pathophysiological mechanisms underlying early pregnancy loss.

In rodent ovaries Kit ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth, and follicle survival. Studies were undertaken to determine whether KITL and KIT carry out similar activities in rabbits. Kitl and Kit mRNA and protein were localized to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary, and growing primary follicles increased significantly in recombinant mouse KITL-treated explants compared with untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse, and these effects were inhibited by coculture with KITL-neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiologic roles of KITL and KIT may differ between species, and this has important implications for the design of in vitro culture systems for folliculogenesis in mammals, including the human.

In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 μg of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1_L:BCL2L1_s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.

Intracytoplasmic sperm injection (ICSI) is a popular method used in assisted conception, and live offspring have been born from a variety of species, including humans. In ICSI, sperm chromatin is introduced into the oocyte together with the acrosome, a structure that does not enter the oocyte during normal fertilization. We compared sperm chromatin remodeling, the potential of embryos to develop in vitro, and DNA synthesis in mouse embryos obtained from in vitro fertilization (IVF) and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development. Sperm chromatin was examined on air-dried, Giemsa-stained preparations at 30-min intervals for up to 4.5 h postfertilization. In all experimental groups, the oocytes underwent activation and formed pronuclei with similar rates. However, the dynamics of sperm chromatin remodeling in ICSI and IVF embryos varied. In ICSI, chromatin remodeling was more asynchronous than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome prior to injection with calcium ionophore A23187 but not with detergent Triton X-100 allowed more synchronous chromatin remodeling, timely DNA synthesis, and good embryo development. Our data have significance for the refinement of the molecular and biologic mechanisms associated with ICSI for current and future applications.

RHO GTPases are key regulators of the actin cytoskeleton and stress fiber formation. In the human uterus, activated RHOA forms a complex with RHO-associated protein kinase (ROCK) which inhibits myosin light chain phosphatase (PPP1R12A), causing a calcium-independent increase in myosin light chain phosphorylation and tension (Ca² sensitization). Recently discovered small GTP binding RND proteins can inhibit RHOA and ROCK interaction to reduce calcium sensitization. Very little is known about the expression of RND proteins in the human uterus. We tested the hypothesis that the uterine quiescence observed during gestation is mediated by an increase in RND protein expression inhibiting RHOA–ROCK-mediated PPP1R12A phosphorylation. Immunohistochemistry and immunoblotting were used to determine RHOA and RND protein expression and localization in nonpregnant, pregnant nonlaboring, and laboring patients at term and patients in spontaneous preterm labor. Changes in protein expression estimated by densitometry between different patient groups were measured. A significant increase of RND2 and RND3 protein expression was observed in pregnant relative to nonpregnant myometrium associated with a loss of PPP1R12A phosphorylation. RND transfected myometrial cells demonstrated a dramatic loss of stress fiber formation and a “rounding” phenotype. RND upregulation in pregnancy may inhibit RHOA–ROCK-mediated increase in calcium sensitization to facilitate the uterine quiescence observed during gestation.

To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes—lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)—with unknown functions in the epididymis. The novel gene, designated Serpina1f (serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except for Lcp1. Testosterone (T) restored these reduced expressions, except for Teddm1; however, this restoration was not observed with 17 beta-estradiol (E2) . Administration of T and E2 combination recovered the Serpina1f mRNA concentration; this recovery was also observed with T alone. However, the recovery of Cuzd1 and Wfdc16 mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed the Cuzd1, Wfdc16, and Serpina1f mRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.

The preoptic/anterior hypothalamic area (POA/AH) is one of the most sexually dimorphic areas of the vertebrate brain and plays a pivotal role in regulating male sexual behavior. Vinclozolin is a fungicide thought to be an environmental antiandrogen, which disrupts masculine sexual behavior when administered to rabbits during development. In this study, we examined several characteristics of the rabbit POA/AH for sexual dimorphism and endocrine disruption by vinclozolin. Pregnant rabbits were dosed orally with vinclozolin (10 mg/kg body weight) or carrot paste vehicle once daily for 6 wk beginning at midgestation and continuing through nursing until Postpartum Week 4. At 6 wk, offspring were perfused with 4% paraformaldehyde and brains processed for immunocytochemical localization of tyrosine hydroxylase, calbindin, gonadotropin-releasing hormone (GnRH), or Nissl stain. There were significant sex differences in the distribution of calbindin in the POA/AH and the size of cells in the dorsal POA/AH (values greater in females than in males), but not in the number or distribution of tyrosine hydroxylase or GnRH neurons. In both sexes, exposure to vinclozolin significantly increased calbindin expression in the ventral POA/AH and significantly decreased number of GnRH neurons selectively in the region of the organum vasculosum of the lamina terminalis (OVLT) but not more caudally in the POA/AH. This is the first documentation of a sexually dimorphic region in the rabbit brain, and further supports the use of this species as a model for studying the influence of vinclozolin on reproductive development with potential application to human systems.

Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.