We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K^b, to the placenta. We show that the transgenic MHC antigen H-2K^b is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K^b-specific maternal B cells in mothers bearing H-2K^b-positive placentas. Our results suggest that transgenic H-2K^b molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female × Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.

Follicles of wave 1 were designated F1, F2, and so forth, according to descending diameter at the expected (F1, ≥8.2 mm) or observed beginning of deviation (Hour 0), as indicated by a reduction in growth rate of F2. During Hours −24 to 0 (experiment 1; n = 34 waves) and Hours −16 to 0 (experiment 2; n = 21), F1 and F2 grew in parallel (no significant differences). During Hours −16 to 0, growth rate was greater (P < 0.05) for F1 (1.4 ± 0.1 mm/16 h) and F2 (1.0 ± 0.1) than for F3 (0.6 ± 0.1) and F4 (0.5 ± 0.1). During Hours 0 to 16, growth rate was greater (P < 0.05) for F1 (1.4 ± 0.2 mm/16 h) than for F2 (0.1 ± 0.1), F3 (0.1 ± 0.1), and F4 (0.1 ± 0.2). In experiment 1, zero, one, two, or three largest follicles were ablated by aspiration of contents at Hour 0 (n = 7/group). For heifers with a single dominant follicle, the dominant follicle formed from the largest retained follicle more often when it was >7.0 mm (14 of 15) than when it was <7.0 mm (0 of 10). When the retained follicles were <7.0 mm, the first follicle to reach 7.0 mm became dominant in seven of eight heifers. Mean hour of observed deviation (occurring after Hour 0 in the ablation groups) increased progressively in groups with increasing number of ablated follicles. Plasma concentrations of FSH for groups with one, two, or three ablated follicles increased to a similar extent between Hours 0 and 12. Results supported the following: 1) during the 24 h before the beginning of deviation, small follicles grew more slowly than large follicles and the largest follicles grew in parallel; 2) after ablation of large follicles, the small retained follicles did not deviate until one reached a diameter characteristic of the beginning of deviation; 3) the potential for dominance at the expected beginning of deviation was greatest for the largest follicle and decreased progressively for the smaller follicles but only when the retained follicles were >7.0 mm; and 4) the three largest subordinate follicles began to deviate simultaneously.

It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca² oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca² oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.

Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.

The zinc finger transcription factor Gata4, is associated with gonadal development in many species. The present study characterizes temporal and spatial localization of Gata4 throughout gonadogenesis in porcine embryos. Immunohistochemical studies illustrated that Gata4 protein is present in the coelomic epithelium prior to histological differentiation of the nascent bipotential gonad, marking the future site of both XX and XY porcine gonads. Many somatic cells of both XX and XY bipotential gonads continue to retain Gata4 immunoreactivity throughout sexual differentiation and subsequent gonadal development. Testicular cords were evident by 26 days postcoitum. Gata4 was present in Sertoli cells, identified by virtue of coexpression with Müllerian inhibiting substance and also interstitial cells including Leydig cells throughout fetal and postnatal life. Many somatic cells of the differentiating ovary including follicular cells also contained Gata4 protein throughout fetal and postnatal life. Gata4 was not present in germ cells, endothelial cells, or other undifferentiated mesenchymal cells of both XX and XY gonads. A population of Gata4-positive cells in the dorsal mesentery was continuous with the coelomic epithelium of the gonad. This localization pattern led to the hypothesis that a subpopulation of somatic cells in the dorsal mesentery moves toward the gonad. An in vitro cell migration assay demonstrated that Gata4-positive cells preferentially migrate toward explanted gonadal tissue, and morphological features of the developing gonad supported this hypothesis. This study illustrates that Gata4 is a very early marker for gonad formation, highlights species differences in temporal and spatial localization patterns, and suggests a potential role for Gata4 in the development of both XX and XY porcine gonads. Further, we suggest that mesenchymal cells of the dorsal mesentery may provide a source of somatic cells that migrate and incorporate into the gonad and contribute to various somatic cell lineages. Overall, the spatial and temporal localization patterns of Gata4 during porcine gonadogenesis implies a much earlier and wider role for Gata4 than previously reported in other species.

Different organs contain fibroblasts with specific features and functions, indicating the complexity of fibroblast biology. In the rat cervical stroma, fibroblasts are preferentially located in the fibrous ring that surrounds the mucous layer. The purpose of this study was to investigate the morphological features and immunophenotype of fibroblastic cells of the uterine cervix in cycling, pregnant, and postpartum rats. Expression of the cytoskeletal proteins desmin, vimentin, and α-smooth muscle actin (α-SMA) were studied by immunohistochemistry. The optical density of immunohistochemical staining was quantified by image analysis. The ultrastructural features of fibroblastic cells were observed under transmission electron microscopy. Cervical fibroblastic cells always expressed vimentin and desmin but never α-SMA. During the first half of pregnancy (Day 5 [D5] to D14), desmin intensity values were similar to those of cycling and postpartum fibroblasts. In contrast, a strong expression of desmin was found from D15 to D22, with maximal expression at term (D23). Immunohistochemical expression for vimentin was constant throughout pregnancy and showed no differences with cycling and postpartum uterine cervices. Stromal cells from cycling and early pregnant rats displayed ultrastructural features characteristic of typical fibroblasts. In contrast, at the end of pregnancy, fibroblasts differentiated and showed increased secretory characteristics, reaching the ultrastructural features of a myofibroblast. Based on the differential expression of desmin and the electron microscopic observations, the foregoing results showed a modulation of the fibroblastic phenotype in the uterine cervix during pregnancy. To our knowledge, this is the first report that addresses the presence of myofibroblasts derived from resident fibroblasts in the fibrous ring of the rat uterine cervix. Fibroblastic-myofibroblastic cell plasticity may have implications in the physiological changes displayed in the uterine cervix during pregnancy, parturition, and postpartum involution.

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3–4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogenous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.

Cystic fibrosis transmembrane regulator (CFTR), multidrug-resistant (MDR)1, and multidrug resistance-associated (MRP) proteins belong to the ATP-binding cassette (ABC) transporter superfamily. A compensatory regulation of MDR1 and CFTR gene expression has been observed in CFTR knockout rodent intestine and in an epithelial cell line of human colon, whereas a high homology and similar anion binding site are shared by MRP and CFTR proteins. To provide better insight into the relationship among the expression behavior in vivo of the three genes in human testis, analysis of MDR1 and MRP gene expression in testicular biopsies was performed and related to the presence of CFTR gene mutations in congenital absence of the vas deferens (CAVD: n = 20) and non-CAVD (n = 30) infertile patients with azoospermia or severe oligozoospermia. A CFTR mutation analysis performed in both groups of patients supported the involvement of CFTR gene mutations in CAVD phenotype (85%) and in defective spermatogenesis (19%). Quantitative reverse transcription-polymerase chain reaction analysis of testicular tissue showed a CFTR-independent MDR1 and MRP gene expression in human testis, suggesting that the mechanisms underlying CFTR gene regulation in testis are different from those in intestine. These findings should contribute to the understanding of patterns of in vivo expression of CFTR, MDR1, and MRP genes in CFTR-related infertility.

The actin cytoskeleton is important for stress fiber formation and contributes to the initiation and maintenance of smooth muscle contraction. To determine if oxytocin and lysophosphatidic acid (LPA) induce stress fiber formation, cultured human myometrial cells were exposed to oxytocin (10⁻⁵ M) or LPA (10⁻⁶ M), and filamentous (F) and globular (G) actin pools were stained with fluorescein isothiocyanate-phalloidin and Texas red DNase I, respectively. The F- to G-actin fluorescent-staining ratio was measured by fluorescence microscopy. Oxytocin and LPA increased stress fiber formation, as indicated by an increase in the F- to G-actin fluorescent-staining ratio. The Rho-kinase inhibitor Y-27632 markedly attenuated this increase. Oxytocin-induced stress fiber formation was completely inhibited in the presence of the oxytocin antagonist compound VI. Tyrosine kinase inhibition with tyrphostin A23 partially blocked the increase induced by oxytocin but had no effect on LPA-induced stress fiber formation. Stress fiber formation was not blocked by pertussis toxin, mitogen-activated protein kinase, or protein kinase C inhibitors. Our results show that human myometrial cells respond to oxytocin and LPA with the formation of stress fibers that may be involved in the maintenance of uterine contractions. Rho-kinase appears to be a key signaling factor in this pathway.

A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogenous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56–70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.

The human seminal vesicles secrete two proteins, semenogelin I and semenogelin II, at very high concentrations. It has previously been shown that the cotton-top tamarin (Sanguinus oedipus), a New World monkey, is lacking the semenogelin II gene. We have now determined the nucleotide sequence of DNA located 5–13 kilobases (kb) downstream of the tamarin semenogelin I gene—a region that in man is occupied by the semenogelin II gene. Two regions with homology to the human semenogelin II gene were identified in the tamarin DNA. The first region, of 3.5 kb, is homologous to DNA upstream of the human gene, and the second region, of 0.6 kb, is mainly derived from the second intron. Between these regions, equivalent to 594 base pairs (bp) upstream of the transcription initiation site to 12 bp downstream of the stop codon in the human semenogelin II gene, the cotton-top tamarin DNA carries a truncated LINE1 repeat. In another set of experiments, the tamarin DNA hybridizing to the mouse semenoclotin gene was investigated. It was concluded that hybridization is with the second intron of the semenoclotin gene, but very likely, the material does not represent a cotton-top tamarin semenoclotin gene. Thus, a mammalian ancestor probably carried a single gene that in the rodent lineage developed into the semenoclotin gene and in the primate lineage into a progenitor of the semenogelin genes.

The increase in LH concentrations at the time of the decrease in FSH concentrations during follicle deviation in mares was studied to determine the role of LH in the production of estradiol and immunoreactive inhibin (ir-inhibin). Ten days after ovulation, all follicles ≥6 mm were ablated, prostaglandin F_2α was given, and either 0 mg (control group, n = 15) or 100 mg of progesterone in safflower oil (treated group, n = 16) was given daily for 14 days, encompassing the day of diameter deviation. The follicular and hormonal data were normalized to the expected day of the beginning of diameter deviation when the largest follicle first reached ≥20 mm (Day 0). The experimentally induced decrease in LH concentrations during follicle deviation beginning on Day −4 delayed and stunted the increase in circulating concentrations of ir-inhibin and estradiol beginning on Days −3 and −1, respectively, but did not alter the predeviation FSH surge and the initiation of diameter deviation between the two largest follicles. Combined for both groups, the interval to the expected day of deviation was 16.6 days after ovulation when the largest follicle was a mean of 21.6 mm. After deviation, the largest follicle started to regress in the treated group beginning on Day 1 and was associated with decreased concentrations of ir-inhibin and estradiol, and increased concentrations of FSH. The negative influence of the dominant follicle on the postdeviation decrease in FSH observed in the control group was alleviated and concentrations resurged in the treated group. Apparently this is the first in vivo evidence that the increase in LH that precedes follicle deviation has a positive effect in supporting the production of inhibin during diameter deviation. It was concluded that the increase in LH concentrations before diameter deviation played a role in the production of estradiol and inhibin by the largest follicle during deviation.

We studied the morphological and quantitative changes in cyclic corpora lutea (CCL) and in CL of pregnancy (CLP) during structural luteolysis. Elimination of CCL takes several cycles, and cell death occurs as successive apoptotic bursts, from 2100 h in proestrus to 1300 h in estrus. Each apoptotic burst determined a 60% decrease in the CL volume and an 80% decrease in the number of steroidogenic cells (SC). All these changes were inhibited by blocking the preovulatory prolactin (PRL) surge with bromocryptine (CB154). Neither apoptotic cells nor changes in the number of SC were found in regressing CLP from Day 21 of pregnancy to Day 2 postpartum, although there was a 50% decrease in the CLP volume and a 30% decrease in the mean cross-sectional area of SC. Treatment with CB154 on the day of parturition did not modify these regressive changes. On Day 5 postpartum, the volume of the CLP and the number of SC were equivalent in lactating rats (showing high PRL concentrations induced by pup suckling) and nonlactating noncycling rats (in which cyclicity and, therefore PRL surges, were blocked by treatment with LHRH antagonist). However, on Day 10 postpartum, the CLP volume and the number of SC were significantly decreased in lactating rats, and apoptotic cells were frequent. In postpartum cycling rats, the CLP did not show apoptotic cells on the day of the second postpartum estrus (on Day 5 postpartum), whereas on the day of the third postpartum estrus (on Day 9 postpartum), apoptotic cells were abundant. These results indicate that PRL does not induce apoptosis in the CLP before Day 5 postpartum and strongly suggest that the proapoptotic effect of PRL is dependent on the degree of differentiation of luteal cells.

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 μM LY294002 or 10 μM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34^cdc2 kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34^cdc2 kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.

The three currently available male contraceptive approaches are 1) the barrier method such as the condom, 2) hormonal methods by disrupting the pituitary-testicular axis so as to impair spermatogenesis, and 3) immunological methods by preparing vaccines against male-specific antigens. We hereby describe an alternative approach in which attachments of developing germ cells onto the seminiferous epithelium are disrupted, thereby inducing their premature release into the tubular lumen. This in turn leads to infertility. A panel of analogues based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid was synthesized. These compounds were subjected to an in vivo screening assay assessing their effects in inducing the expression of testin, a testicular marker whose expression correlates with the integrity of Sertoli-germ cell junctions. An induction of testin expression in the testis signifies a disruption of Sertoli-germ cell junctions that is followed by depletion of germ cells from the seminiferous epithelium. Two compounds, namely 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF-2785), were identified that caused detachment of germ cells, in particular round and elongated spermatids, from the epithelium inducing their premature release into the tubular lumen as confirmed by histological analysis. Adult rats receiving several oral doses of either one of these compounds became infertile within 3–7 wk after the epididymal sperm reserve was exhausted. Depending on the dosing of the administered compound, rats became infertile for 4–14 wk before their fertility gradually bounced back, illustrating the reversibility and efficacy of these new compounds. Also, these compounds did not appear to impair the hypothalamus-pituitary-testicular axis because the serum levels of LH, FSH, and testosterone of the treated animals did not change significantly when compared to control rats. In addition, results of serum microchemistry illustrate that liver and kidney function was not affected in animals treated with both compounds.

The primary aim of this study was to establish a flow cytometric technique for determining the capacitation status of stallion spermatozoa. To this end, a flow cytometric technique that demonstrates changes in plasma membrane fluidity; namely, merocyanine 540 staining, was compared with the more conventional Ca² -dependent fluorescence microscopic technique, chlortetracycline (CTC) staining, for assessing capacitation status. In addition, the effect of bicarbonate/CO₂ on the progress of capacitation and the acrosome reaction (AR) and on temporal changes in sperm motility, with particular regard to hyperactivation, was analyzed. For the study, fresh semen was washed and then incubated for 5 h in bicarbonate-containing or bicarbonate-free medium, with or without Ca² ionophore to induce the AR, and at intervals during incubation aliquots were taken and analyzed for capacitation and acrosome status. The AR was assessed using both the CTC and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) staining techniques with similar results. In brief, it was found that merocyanine 540 detects capacitation-related changes much earlier than CTC does (0.5 h versus ∼3 h), and that flow cytometry for evaluation of capacitation and AR was a quicker (10 sec per sample) and more accurate (10 000 cells counted) technique than fluorescence microscopy. Furthermore, it was observed that Ca² ionophore could not induce the AR in the absence of bicarbonate, but that the ionophore synergized the bicarbonate-mediated induction of the AR as detected by CTC (although it was not significant when evaluated using FITC-PNA). The percentage of hyperactive sperm in each sample was not affected by time of incubation under the experimental conditions studied. In conclusion, merocyanine 540 staining is a better method than CTC staining for evaluating the early events of capacitation for stallion spermatozoa incubated in vitro. Furthermore, bicarbonate sperm activation clearly plays a vital role in the induction of the AR in stallion spermatozoa.

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17α-hydroxylase, cytochrome P450 aromatase, 3β-hydroxysteroid dehydrogenase Δ⁴,Δ⁵ isomerase, LH, and FSH receptors and estrogen receptor-β in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17β, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17β (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17β concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-β mRNAs than other groups. In summary, increased expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17β concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNFα) and transforming growth factor alpha (TGFα) have been suggested. TGFα stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNFα and its possible interaction with TGFα in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNFα and TGFα interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGFα (0.1 or 10 ng/ml), but not TNFα (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin β₃ subunit was also stimulated by TGFα in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNFα in the presence of maximal stimulatory concentrations of TGFα potentiated this response. TGFα increased both F5,6 and F1 granulosa cell [³H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNFα had no effect on [³H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNFα and TGFα than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGFα-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNFα alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGFα but not of TNFα. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNFα and TGFα was necrosis and not apoptosis. The current study demonstrates that TNFα and TGFα interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNFα; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.

The MSJ-1 gene encodes a murine DnaJ homologue that is expressed specifically in adult testis. DnaJ proteins act as cochaperones of Hsp70 proteins in promoting diverse cellular functions. In this study we used recombinant MSJ-1 proteins to produce MSJ-1 antiserum and to carry out in vitro binding assays. In a wide immunoscreening of mouse tissues, affinity-purified MSJ-1 antibodies recognize a unique protein of 30 kDa in male germ cells only. MSJ-1 is able to interact with the testis-specific Hsp70-2 protein and can be coimmunoprecipitated with Hsp70-2 from spermatogenic cells; binding of these two chaperones is consistent with the presence of a third component, which is so far unknown. MSJ-1 is weakly detected in early round spermatids, and its protein content increases in cytodifferentiating spermatids where it colocalizes with the developing acrosome and their postnuclear region. Hsp70-2, which is known to be highly expressed in meiotic cells, shows a subcellular localization in late differentiating spermatids that overlaps that of MSJ-1. MSJ-1 is also maintained in testicular and epididymal spermatozoa, where it sharply demarcates into two distinct cell areas; the outer surface of the acrosomal vesicle, and the centrosomal area. On the whole, our findings are consistent with a role for MSJ-1 in acrosome formation and centrosome adjustment during spermatid development, whereas its presence in mature spermatozoa suggests a special function during fertilization, shortly afterward, or both.

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-β receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-γ1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI₂). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA₂ inhibitor, and in the absence of Ca² . U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI₂ biosynthesis. PDGF-BB also increased cell proliferation and [³H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-α-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12–18). However, bikunin unbound to the inter-α-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.

The purpose of this study is to determine whether inducible nitric oxide synthase (iNOS) is involved in the pathogenesis of testicular ischemia-reperfusion (I/R) injury in association with germ cell death, through either necrosis or apoptosis. Western blot analysis showed that iNOS expression was markedly increased 1 h after ischemia, and was accompanied by a huge nitric oxide (NO) production, as measured by the Griess method, with a peak at 48 h of reperfusion. Immunohistochemistry showed that iNOS was expressed predominantly in the macrophage-like cells infiltrated in the interstitial tissues of the testis. Intraperitoneal injection of aminoguanidine (AMG) (400 mg/day), the inhibitor of iNOS, reduced NO production by 57.7% at 96 h of reperfusion. Calpain activation and proteolysis of α-fodrin induced by I/R were inhibited by AMG. Germ cell apoptosis was demonstrated by in situ TUNEL and DNA fragmentation on agarose gel electrophoresis. Germ cell apoptosis was maximally induced at 24 h of reperfusion, and was not inhibited by AMG. NO produced by iNOS in the delayed phase of reperfusion promoted α-fodrin proteolysis, which is closely associated with necrosis. Inducible NOS inhibition combined with calpain inhibition may improve impaired spermatogenesis after testicular torsion.

In the highly organized and complex process of mammalian spermatogenesis, the development of an undifferentiated diploid germ cell into a fully differentiated and mature spermatozoon is orchestrated in a time frame unique for each species including man. If the various hormonal signals including environmental cues that play a critical part in initiating these events are not properly executed, various deficiencies including delay in sexual maturity or puberty are likely. In this study we have followed testicular development and spermatogenesis in the FSH receptor knockout (FORKO) mice from Day 7 onward by using histology and quantitative DNA flow cytometry. The drastic reduction in testicular weight and shrinkage of seminiferous tubules that occurred at this early age persisted into the adult stage in the FORKOs, suggesting inhibition of the initial developmental processes. The round spermatids that were clearly abundant on Day 21 in the wild-type and heterozygous males were few and present only in some tubules of the FORKOs. There were no elongated spermatids in FORKO males on Day 35. The sperm produced by Day 49 FORKOs were already aberrant, a feature that persisted into adulthood in these animals. As all these changes occurred in a background of normal circulating testosterone levels, we may conclude that the delay in testicular development is a consequence of the loss of FSH-receptor signaling. The delay in sexual maturity of FORKOs was accompanied by reduction in fertility as evidenced by mating studies. Based on these data we suggest that the FORKO mouse might be a useful experimental model to define the molecular mechanisms that underlie the delay in puberty.

Male juvenile spermatogonial depletion (jsd/jsd) mice are sterile because of a failure of spermatogonial differentiation. We have previously reported the recovery of spermatogonial differentiation by suppressing the levels of gonadotropins and testosterone with Nal-Glu, a GnRH antagonist. To determine whether suppression of testosterone or the gonadotropins was responsible for spermatogenic recovery, we examined the effect of supplementation of LH or FSH along with Nal-Glu treatment. Systemic administration of flutamide, an androgen receptor antagonist, was also examined. LH supplementation elevated both serum and intratesticular testosterone levels and suppressed the recovery of spermatogonial differentiation in a dose-dependent manner. Supplementation with FSH did not affect either testosterone levels or spermatogonial differentiation. Furthermore, the mice treated with flutamide showed some recovery of spermatogonial differentiation. The overall findings revealed that testosterone action mediated by androgen receptors suppressed the spermatogonial differentiation in jsd/jsd mice and suggested that spermatogonial differentiation in the jsd mutant is highly sensitive to testosterone suppression.

In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK₁ receptor (NK₁R), the tachykinin NK₂ receptor (NK₂R), and the tachykinin NK₃ receptor (NK₃R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK₁R, the NK₂R, or the NK₃R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK₁R and NK₃R varied along the course of pregnancy and at postpartum. Uterine NK₂R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK₂ receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 μM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.

In the musk shrew, Suncus murinus (and other shrews), the cumulus oophorus is ovulated as a discrete, compact, matrix-free ball of cells linked by specialized junctions. In examining how they penetrate the cumulus, Suncus spermatozoa were observed to first bind consistently by the ventral face over the acrosomal region to the exposed smooth surface of a peripheral cumulus cell. This was apparently followed by point fusions between the plasma and outer acrosomal membranes. Thereafter, spermatozoa without acrosomes were observed within cumulus cells that displayed signs of necrosis, as did some radially neighboring cumulus cells linked by zona adherens and gap junctions. Eventually, penetration of spermatozoa as far as the perizonal space around the zona pellucida left linear tracks of locally necrotic cells flanked by normal cumulus cells. Based on these and previous observations, we conclude that the acrosome reaction in Suncus is always induced by cumulus cells, and that reacted spermatozoa penetrate the cumulus by selective invasion and killing of cumulus cells along a linear track. Loss of the acrosome also exposes an apical body/perforatorium that is covered with barbs that appear to assist reacted fertilizing spermatozoa in binding to the zona pellucida. Because fertilized eggs displayed no other spermatozoa within or bound to the zona, an efficient block to polyspermy must prevent such binding of additional spermatozoa.

The reproductive health risks related to exposure to persistent organic pollutants in the environment remain controversial. This debate is partly because most studies have investigated only one or two chemicals at a time, whereas populations are exposed to a large spectrum of persistent chemicals in their environment. Using the pig as a toxicological model, we hypothesized that exposing immature cumulus-oocyte complexes to an organochlorine mixture during in vitro maturation (IVM) would adversely affect oocyte maturation, fertilization, and subsequent embryo development. This organochlorine mixture mimics that which contaminates the Arctic marine food chain. Cumulus-oocyte complexes were cultured in IVM medium containing increasing concentrations of the organochlorine mixture, similar to that found in women of highly exposed populations. Organochlorines reduced the quality of cumulus expansion and the viability of cumulus cells in a dose-response manner. The proportion of apoptotic cumulus cells also increased due to organochlorine exposure. Half of the oocytes were fixed after insemination, and the remainders were cultured for 8 days. Concentrations of organochlorines did not affect the rates of oocyte degeneration, sperm penetration, and development to morula. However, incidence of incompletely matured oocytes increased and polyspermy rate decreased, both in a dose-response manner with increasing organochlorine concentrations. Blastocyst formation and number of cells per blastocyst declined with organochlorine concentration. Exposing porcine cumulus-oocyte complexes to an environmentally pertinent organochlorine mixture during IVM disturbs oocyte development, supporting recent concerns that such pollutants harm reproductive health in humans and other mammalian species.

Stimulation of mouse GnRH receptor promoter by a GnRH agonist (Buserelin), or by a cAMP analogue, significantly increased reporter (luciferase) activity. Overexpression of Raf-1, ERK1, or ERK2 partially blocked Buserelin-stimulated luciferase activity. In contrast, treatment with a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 98059) activated basal and Buserelin-stimulated luciferase activity in a dose-dependent manner. Transient transfection of the deleted cAMP response element expression vector followed by pretreatment with PD98059 prior to Buserelin stimulation showed that the transcriptional response was decreased compared to wild-type promoter. A gel-mobility shift assay using a probe containing the cAMP response element showed the presence of two specific protein-DNA complexes that contain one or more members of the cAMP responsive element-binding (CREB) protein family. These results suggest that cAMP and CREB participate in the GnRH activation of GnRH receptor promoter activity and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRH receptor transcriptional activity.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and the maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight regarding the regulation of Sertoli cell differentiation. The optimal activation of the mouse transferrin promoter (mTf) by FSH requires the synergistic actions of the cAMP response element-binding protein (CREB) binding to the cAMP response element-like proximal region II (PRII) and the basic helix-loop-helix (bHLH) binding to the E-box. Proximal region II alone is sufficient for cAMP-mediated activation. The proximity of the PRII and E-box (220 base pairs apart) suggests the possibility of interaction between CREB and bHLH proteins. Such an interaction can be mediated by transcriptional integrators such as CREB-binding protein (CBP) and/or p300 and may stabilize the binding of trans-acting factors to their respective cis-elements. Such an interaction may also provide a mechanism for cell-specific promoter activation. The hypothesis tested in this study was that CBP/p300 is required for the synergistic activation of the transferrin promoter involving PRII and E-box through the formation of a ternary complex. In the Sertoli cells, both CBP and p300 proteins are expressed. The effect of CBP/p300 on transferrin promoter activation and, hence, Sertoli cell function was studied by using antisense oligonucleotides (AS-oligo). In the presence of CBP/p300 AS-oligo, activity of the FSH-induced mTf-chloramphenicol acetyl transferase (CAT) was significantly lower as compared to the respective controls. Interestingly, AS-oligo had no effect on cAMP-induced activation of the transferrin promoter reporter construct (mTf-CAT). Mutations in the E-box (EB*) significantly reduced the FSH response. The presence of AS-oligo had no further effect on the FSH-mediated activation of the EB*-mTf-CAT construct but reduced cAMP-mediated activation. Mutations in the CRE-like PRII (PRII*) also significantly reduced the FSH response. Activation of the PRII*-mTf-CAT in response to cAMP was completely abolished. The presence of AS-oligo had no further effect on the FSH- or cAMP-mediated activation of the PRII*-mTf-CAT construct. In Sertoli cells, CBP/p300 was coimmunoprecipitated with CREB and the bHLH protein E47. These observations suggest that CBP/p300 appears to be involved in regulating FSH-mediated activation of the transferrin promoter by linking bHLH and CREB activities.

In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.

The hypothesis was that long-term treatment of cattle with estradiol (E₂) and bovine somatotropin (bST) would alter the earliest stages of folliculogenesis. Nonlactating Holstein cows (n = 26) were treated in a 2 × 2 arrangement with E₂ (2 × 24 mg implants, 67.1 ± 1.4 days) and bST (Posilac, 63.6 ± 1.5 days). At Day 67 ± 1.3, one ovary was removed for morphometric and immunohistochemical analysis. For each ovary, 388 ± 38 microscopic fields (2 × 2 mm) were examined and follicles within each field were classified by histological stage. Fields that contained no follicles were classified as empty. Empty fields (n = 100 per ovary) were further classified as containing no evidence of follicles or containing atretic remnants of follicles. Approximately 30 4-μm sections per ovary were stained for proliferating cell nuclear antigen (PCNA), and 150 fields per ovary were evaluated. Additional sections (n = 10 per ovary) were assessed immunohistochemically for apoptosis, and fluorescence intensity was determined for each follicle. Treatment with bST significantly decreased percentage of empty fields containing atretic remnants. Treatment with E₂ induced activation of follicles as shown by a decrease in percentage of primordial follicles and an increase in percentage of primary follicles as determined by PCNA staining. At the primary follicle stage the combination of bST E₂ decreased apoptosis as shown by decreased fluorescence intensity. Thus, E₂ induced activation of follicles, bST enhanced survival, and the combination lowered atresia.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N¹-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

To study the regulation of fetal testicular steroidogenesis in the rat, we examined effects of members of the natriuretic peptide family, that is, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), on testosterone production of dispersed Leydig cells of rat fetuses at Embryonic Day (E) 18.5. All three peptides stimulated testosterone production, with significant effect at concentrations ≥1 × 10⁻⁸ mol/L of ANP, ≥1 × 10⁻⁹ mol/L of BNP, and ≥1 × 10⁻⁶ mol/L of CNP. Likewise, receptors for all three peptides (i.e., NPR-A, NPR-B, and NPR-C) were expressed in the fetal testis as early as E15.5. The natriuretic peptides had no effect on cAMP production by fetal Leydig cells. When tested in combination with two other peptides previously shown to stimulate fetal testicular steroidogenesis, vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27), the combined effects did not differ significantly from the maximum effect with any one of the peptides alone. In conclusion, our present findings provide both functional and molecular evidences for NPR-A, NPR-B, and NPR-C in the fetal testis. Because ANP has previously been detected in fetal plasma and we now demonstrate the expression of BNP and CNP in fetal testes, these findings indicate involvement of the natriuretic peptides in endocrine and paracrine regulation during the early phase of fetal testicular steroidogenesis at E15.5–19.5 (i.e., before the onset of pituitary LH secretion).

The present work reports on testicular hormone-sensitive lipase (HSL), the biological significance of which has been documented in male fertility. The HSL protein levels and enzymatic activity were measured, respectively, by densitometry of immunoreactive bands in Western blots, performed with antibodies against recombinant rat HSL, and by spectrophotometry in seminiferous tubules (STf) and interstitial tissue (ITf) enriched fractions generated from neonatal, pubertal, and adult guinea pig testes. In addition, HSL was studied in subcellular fractions obtained from STf isolated from adult testes and in epididymal spermatozoa (Spz). A 104-kDa HSL protein was detected in STf and ITf, the expression and activity of which increased with testicular development. Three immunoreactive bands of 104, 110, and 120 kDa were detected in the lysosomal subfraction, and two bands of 104 and 120 kDa were detected in Spz. The HSL activity was positively correlated with free (FC) and esterified (EC) cholesterol ratios in STf and ITf, but not with triglyceride (TG) levels, during testicular development. Immunolabeling localized HSL to elongated spermatids and Sertoli cells, where its distribution was stage-dependent, and within the cells lining the excurrent ducts of the testis. The findings of the 104- and 120-kDa HSL immunoreactive bands and of HSL activity in Spz as well, as the detection of the 104-, 110-, and 120-kDa immunoreactive bands in lysosomes, suggest that part of HSL may originate from germ cells and be imported in Sertoli cells. The HSL protein levels and enzymatic activity in ITf and STf were positively correlated with serum testosterone levels during development. To the best of our knowledge, this study is the first to contribute insights regarding the impact of HSL on FC:EC cholesterol ratios and TG levels in the interstitial tissue and tubules in relation to serum testosterone levels during postnatal development, and regarding the immunolocalization of the enzyme in regions of the male gamete consistent with spermatozoa-oocyte interaction.

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.

2-Methoxyestradiol (2-ME) is an estradiol metabolite with antiangiogenic and antitumor activity. It is formed by granulosa cell (GC) catechol-O-methyltransferase activity and is present in the normal follicle at high concentrations. In this unique microenvironment, it may regulate selected cell types via autocrine and/or paracrine action. To assess the possibility that 2-ME or estradiol might exert differential mitotic and/or apoptotic effects on endothelial cells and GCs, we compared their actions on primary cultures of hormone- and/or growth factor-stimulated porcine GCs (pGCs) as well as two types of endothelial cells, primary cultures of porcine endothelial cells (pECs), and a spontaneously transformed rabbit endothelial vascular cell (REVC) line. The 2-ME, but not estradiol, dose dependently suppressed tritiated thymidine (³H-T) incorporation into epidermal growth factor (EGF)-stimulated REVCs and EGF/insulin (INS)-stimulated pECs. In contrast, 2-ME did not attenuate incorporation in FSH/INS-stimulated pGCs. It reduced incorporation by approximately 50% in EGF/INS-stimulated pGCs, indicating that responsiveness to 2-ME in normal cells can be modulated by hormone and growth factor treatment. Estradiol was not antimitotic to pGCs. As indicated by 4′,6-diamido-2-phenylindole hydrochloride nuclear staining, estradiol was nonapoptotic in either cell type, and 2-ME significantly increased apoptosis of REVCs, but not of pGCs. In a cell migration assay, REVC movement was attenuated by 2-ME, but not by estradiol. In summary, the results show that antimitotic as well as proapoptotic responses to 2-ME vary with cell type and, in the case of pGC antimitotic activity, with the regulatory microenvironment. Thus, they provide a rationale for autocrine and/or paracrine action of 2-ME at its site of production in vivo, and they strongly support the concept of 2-ME as a candidate ovarian angiogenesis inhibitor.

This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60^src from the Rous Sarcoma Virus When subjected to two-dimensional electrophoresis, this 80-kDa protein migrated as several isoforms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequence analysis of a peptide obtained following trypsin digestion of the bull sperm protein showed homology to the PH-20/hyaluronidase precursor sperm protein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased immunolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react lost their labeling almost completely. As for the PH-20 protein, the 80-kDa bull sperm protein possesses a hyaluronidase activity that is higher at pH 7.0 than at pH 4.0 in an in-gel assay. Unlike what has been observed in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C or with trypsin. However, this protein was not sedimented by a 100 000 × g centrifugation after nitrogen cavitation, which suggests that the 80-kDa protein is loosely attached to the sperm membrane by a yet-unknown mechanism. These results suggest that the 80-kDa bull sperm protein shares many homologies with the sperm PH-20 protein reported in the literature and, most likely, is the bull sperm homologue of the PH-20.