Leptin is a polypeptide hormone that aids in the regulation of body weight and energy homeostasis and is linked to a variety of reproductive processes in both animals and humans. Thus, leptin may help regulate ovarian development and steroidogenesis and serve as either a primary signal initiating puberty or as a permissive regulator of sexual maturation. Perhaps significantly, peripheral leptin concentrations, adjusted for adiposity, are dramatically higher in females than in males throughout life. During primate pregnancy, maternal levels that arise from adipose stores and perhaps the placenta increase with advancing gestational age. Proposed physiological roles for leptin in pregnancy include the regulation of conceptus growth and development, fetal/placental angiogenesis, embryonic hematopoiesis, and hormone biosynthesis within the maternal-fetoplacental unit. The specific localization of both leptin and its receptor in the syncytiotrophoblast implies autocrine and/or paracrine relationships in this endocrinologically active tissue. Interactions of leptin with mechanisms regulating pre-eclampsia and maternal diabetes have also been suggested. Collectively, therefore, reports suggest that a better understanding of the regulation of leptin and its role(s) throughout gestation may eventually impact those causes of human perinatal morbidity and mortality that are exacerbated by intrauterine growth retardation, macrosomia, placental insufficiency, or prematurity.

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F_2α, the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF_2α on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF_2α. Enzymatic activity of PGDH, measured by rate of conversion of PGF_2α to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0.01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 ± 4 and 44 ± 28 amol/μg poly(A) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF_2α, early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF_2α.

The testis-specific histone H1t gene is expressed only in pachytene primary spermatocytes during spermatogenesis. There is a correlation between the specific binding of testis nuclear proteins to a rat histone H1t promoter sequence, designated the H1t/TE element, and the onset of transcription in primary spermatocytes. Our laboratory has shown that mice bearing the rat gene with a deletion of the TE promoter element and replacement with a heterologous stuffer DNA fragment fail to express the rat H1t transgene in any tissue. In this study we report that five CpGs located within the H1t proximal promoter, including two CpGs located within the essential TE promoter element, contain unmethylated cytosines in vivo in genomic DNA derived from primary spermatocytes where the H1t gene is expressed. All seven CpGs are hypermethylated in vivo in genomic DNA derived from liver cells where gene expression is repressed. Further, in vitro methylation of an H1t promoter-driven reporter plasmid markedly reduced expression in a transient transfection assay system. These results suggest that cytosine methylation may contribute to the transcriptional silencing of the testis-specific histone H1t gene in nonexpressing tissues such as liver.

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.

We previously proposed that an endothelin-angiotensin-atrial natriuretic peptide system may contribute to inducing ovulation of mature bovine follicles by modulating follicular secretion of steroids and prostaglandins (PGs). Thus, this study aimed to determine the real-time changes in the local release of angiotensin II (Ang II), endothelin (ET), atrial natriuretic peptide (ANP), PGF_2α, and steroid hormones from bovine mature follicles during the periovulatory period in vivo. Seven cows were treated for superovulation using FSH and PGF_2α injections. Two dialysis capillary membranes per follicle were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system (MDS). Fractions of the perfusate were collected from Day −1 (Day 0 = LH surge) to Day 3. Five out of seven treated cows were normally ovulated, and the newly formed corpora lutea were observed at the end of the experiment. In these five ovulated cows, the release of estradiol, androstenedione, and progesterone in the theca layer increased (P < 0.05) synchronously with the LH surge. Acute increases in PGF_2α and Ang II concentrations in the ovarian venous plasma (OVP) were observed at 24–48 h after the peak of the LH surge, when multiple ovulations were expected to occur. The follicular Ang II release was low during the pre-LH surge period and rose (P < 0.05) at the beginning of the increase in the LH surge. On the other hand, ET-1 release dropped (P < 0.05) when plasma LH started to increase. However, no clear changes in ANP concentration in the MDS perfusate and plasma were observed. The above local changes in Ang II, PGF_2α, as well as steroid hormones were not observed in cows (n = 2) that did not show an LH surge and ovulation. The present results demonstrate for the first time the local release of Ang II, ET-1, and ANP from the bovine mature follicle in real-time in vivo and show that Ang II and PGF_2α concentrations in the OVP acutely increase around the time of ovulation. The overall results support the concept of a local functional ET-Ang-ANP system in the bovine mature follicle that may be involved in the ovulatory process.

Pregnancy can influence both the resting membrane potential and the ion channel composition of the uterine myometrium. Calcium flux is essential for excitation-contraction coupling in pregnant uterus. The uterine L-type calcium channel is an important component in mediating calcium flux and is purported to play a role in parturition. This study was undertaken to characterize gestational changes in 1) the uterine contractile response to the L-type calcium channel agonist, Bay K 8644; 2) the mRNA expression of channel subunits by semiquantitative reverse transcriptase-polymerase chain reaction; and 3) estimate channel protein levels by measuring ³H-isradipine binding at the dihydropyridine binding site of the α_1c subunit utilizing saturation binding methods. Sensitivity to Bay K 8644 increases beginning at 0.8 of gestation and persists through term. The change in sensitivity is coincident with an increased mRNA expression of the α_1c and β₂ subunits but with the least detectable amounts of isradipine binding. The expressed α_1c transcript represents a novel structural variant with a 118-amino acid deletion in the III–IV linker and repeats IVS1–S3 of the protein sequence. The guinea pig uterine L-type calcium channel activity is highly regulated through gestation, but the regulation of mRNA expression may be different from regulation of protein levels, estimated by isradipine binding. The up-regulation of function, α_1c subunit mRNA expression, and isradipine binding at term gestation are consistent with a role for this ion channel in parturition.

Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKCα by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKCα, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase Cα belongs to the Ca² -dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca² in the incubation medium. Protein kinase Cα is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKCα and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKCα and the PKCα-PLD1 complex decomposes after treatment of the cells with LPA or 12-O-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKCα to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKCα and PLD1 activation during the sperm acrosome reaction is suggested.

Our previous studies have shown that bovine granulosa cells cultured in basal media supplemented with 5% fetal bovine serum (BM-FBS) are resistant to apoptosis induced by recombinant Fas ligand (FasL) unless pretreated with interferon-γ (IFN). Experiments were conducted to test the hypothesis that serum and growth factors alter the susceptibility of granulosa cells to FasL-induced apoptosis. Granulosa cells were cultured in BM-FBS, BM containing insulin, transferrin, selenium, and BSA (BM-ITS), and in BM-ITS supplemented with insulin-like growth factor-I (IGF). Cells were susceptible to FasL-induced killing in BM-ITS (27% killing) but were resistant in BM-FBS and in BM-ITS containing IGF (P < 0.05 vs. killing in BM-ITS). Exposure of phosphatidylserine residues on the outer cell membrane, an early marker of apoptosis, was stimulated by FasL and prevented in the presence of IGF. Neutralization of IGF activity in serum with IGF binding protein 3 reduced the protective effect of FBS on FasL-induced killing (P < 0.05), suggesting that IGF is an inhibitory component in FBS. Cotreatment with IFN overcame the inhibitory effects of serum and IGF on FasL-induced killing (31% and 29% killing, respectively, P > 0.05), but IFN did not potentiate killing of cells cultured in BM-ITS. IFN increased expression of Fas antigen (Fas, the receptor for FasL) mRNA five- to sevenfold (P < 0.05) and increased immunostaining for Fas protein similarly in all types of media. Addition of the growth factors epidermal growth factor or basic fibroblast growth factor to BM-ITS also inhibited FasL-induced killing (P < 0.05), whereas keratinocyte growth factor, transforming growth factor, platelet-derived growth factor, FSH, and LH had no effect. In summary, FasL-induced killing is inhibited by FBS and certain growth factors. IFN increased expression of Fas similarly in all types of media but was required for FasL-induced killing only in BM containing FBS or IGF. Therefore, modulation of responsiveness to FasL-induced apoptosis by growth factors and IFN is not directly related to the level of Fas expression.

In vitro data have indicated that nitric oxide (NO) inhibits Leydig cell testosterone production, suggesting that NO may play a role in the suppression of steroidogenesis and spermatogenic function during inflammation. Consequently, we investigated expression of the inflammation-inducible isoform of NO synthase (iNOS) in the inflamed adult rat testis and the ability of a broad-spectrum inhibitor of NO production, l-nitro-l-arginine methyl ester, to prevent Leydig cell dysfunction during inflammation. Unexpectedly, immunohistochemical and mRNA data established that iNOS is expressed constitutively in Leydig cells and in a stage-specific manner in Sertoli, peritubular, and spermatogenic cells in the normal testis. Expression was increased in a dose-dependent manner in all these cell types during lipopolysaccharide (LPS)-induced inflammation. In noninflamed testes, treatment with the NO synthase inhibitor reduced testicular interstitial fluid formation and testosterone production without any effect on serum LH levels. Administration of the inhibitor did not prevent the suppression of testicular interstitial fluid and testosterone production that occurs within 6 h after LPS treatment. Collectively, these data indicate a novel role for iNOS in autocrine or paracrine regulation of the testicular vasculature, Leydig cell steroidogenesis, and spermatogenesis in the normal testis. The data suggest that increased NO is not the major cause of acute Leydig cell dysfunction in the LPS-treated inflammation model, although a role for NO in this process cannot be excluded, particularly at other time points. Moreover, up-regulation of iNOS may contribute to the seminiferous epithelium damage caused by LPS-induced inflammation.

New rat models are being developed at an exponential rate, making improved methods to cryopreserve rat embryos extremely important. However, cryopreservation of rat embryos has proven to be difficult and expensive. In this study, a series of experiments was performed to characterize the fundamental cryobiology of rat fertilized 1-cell embryos (zygotes) and to investigate the effects of different cryoprotective agents (CPAs) and two different plunging temperatures (T_p) on post-thaw survival of embryos from three genetic backgrounds. In the initial experiments, information on the fundamental cryobiology of rat zygotes was determined, including 1) the hydraulic conductivity in the presence of CPAs (L_p), 2) the cryoprotectant permeability (P_CPA), 3) the reflection coefficient (σ), and 4) the activation energies for these parameters. P_CPA values were determined for the CPAs, ethylene glycol (EG), dimethyl sulfoxide (DMSO), and propylene glycol (PG). Using this information, a cryopreservation method was developed and the cryosurvival and fetal development of Sprague-Dawley zygotes cryopreserved in either EG, DMSO, or PG and plunged at either −30 or −80°C, were assessed. The highest fetal developmental rates were obtained using a T_p of −30°C and EG (61.2% ± 2.4%), which was not different (P > 0.05) from nonfrozen control zygotes (54.6% ± 3.0%).

Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (T_p), followed by plunging into liquid nitrogen (LN₂). If the slow cooling step allowed a critical concentration ([CPA]_c) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5°C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.

The present study was designed to 1) investigate whether apoptosis is responsible for the atresia of nonovulatory dominant follicle (DF), 2) to determine if atresia of a nonovulatory DF is associated with alterations in Bcl-2 and Bax expression, 3) to test whether progesterone P₄ has a direct effect on apoptosis in bovine follicles, and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). In experiment 1, 16 cycling cows received a norgestomet ear implant at proestrus (Day 1) for 9 days to mimic the subluteal phase. The cows were assigned either to a control (n = 4) or P₄-treated groups (n = 12). Injections of P₄ (150 mg, i.m.) were given on Day 3 (n = 4); on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Controls received injections of corn oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 to recover DFs from cows that had been treated with P₄ for 24, 48, and 72 h, respectively. DFs in the control group were collected on Day 6. The onset of atresia of DFs was assessed morphologically by ultrasound to determine DF diameters, histologically by light microscopic inspection of tissue sections, and functionally by quantification of follicular fluid steroid hormone levels. Apoptosis was detected by DNA analysis and in situ TUNEL labeling. Expression of Bcl-2 and Bax proteins was examined by Western blot analysis. The earliest signs of atresia were detected 24 h after P₄ injection as evidenced by decreased diameter, degeneration and detachment of granulosa cells (GCs) from the basal lamina, and a dramatically reduced ratio of estrogen to P₄. Electrophoretic analysis of DNA extracted from DFs of cows treated with P₄ for 24 h revealed a distinct ladder pattern of DNA fragments. In contrast, this pattern was not obvious in DFs from control cows. Similar results were also obtained from TUNEL analysis of DFs. Furthermore, both Bcl-2 and Bax were found to be present in all DFs; however, the ratio of Bcl-2 and Bax protein levels was significantly reduced by 24 h of P₄ treatment compared with DFs from the control group (P < 0.05). Experiment 2 investigated the direct effect of P₄ (4 ng/ml) on apoptosis of cultured GCs using ovaries obtained from a local slaughterhouse. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large) was studied. No increase in apoptotic DNA fragments was detected in GCs treated with P₄. The ratio of Bcl-2 and Bax protein levels was variable in small follicles; however, Bax protein level was always relatively higher than that of Bcl-2 in medium and large follicles. In conclusion, our study suggests that apoptosis is the mechanism that underlies the atresia of nonovulatory DFs that develops during the luteal phase of bovine estrous cycle.

Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.

The action of steroid hormones is primarily mediated via a process that involves hormone binding to specific receptors in target cells, which leads to transcriptional activation of steroid-responsive genes and, subsequently, to a modification of cellular responses. The aim of the present study was to obtain information about the dynamics of the two types of estrogen receptors (ERs), α and β, by comparing their concentration and distribution in the reproductive tract of the rat during the estrous cycle. Twenty-four 55- to 60-day-old female Sprague-Dawley rats were used. The stage of estrous cycle was determined by vaginal smear. ERα was the dominating subtype in uterus, oviduct, and cervix/vagina, with the distribution varying in stroma and epithelium during the estrous cycle. A low level of ERα mRNA was observed in ovarian stromal cells, with some scattered positive cells found among granulosa cells. ERβ expression was observed in the different compartments of uterus and cervix/vagina, but cyclic variation during the estrous cycle was less evident than that of ERα. Only a few scattered cells that contained ERβ mRNA were observed in oviduct. ERβ mRNA was highly expressed in granulosa cells of developing follicles, with a weaker hybridization signal in new corpora lutea. Immunohistochemistry showed that protein levels of ERα and ERβ have distinct specificity for tissues and cell types, similar to their respective levels of mRNA, as assessed by in situ hybridization. The precise physiological function and importance of ERβ is still unclear. The relative physiological and pathological function of each ER subtype in the female reproductive tract remains to be further evaluated.

Clusterin is a ubiquitous glycoprotein that is promiscuously expressed at a low basal level but can be highly induced by a variety of stress conditions. In contrast, in some secretory cells associated with tissue-fluid interfaces such as the Sertoli cells in the testis, clusterin demonstrates high constitutive expression. In this study, we address the mechanisms that regulate the constitutive expression of the clusterin gene by using primary cultures of immature rat Sertoli cells. We have identified a region of the rat clusterin gene promoter that activated transcription only in Sertoli cells and that mapped between positions −426 and −311. Sequence analysis of this region revealed a high concentration of potential regulatory elements. Using gel-shift assays combined with hydroxyl radical footprinting, we identified the elements recognized by the Sertoli cell nuclear factors. Comparison of the interactions with this region of the nuclear factors from different cell types demonstrated that recognition of the core-enhancer element is specific for the Sertoli cells, and in vitro, the core region was recognized by the transcription factor CBF. Transient transfections showed that a core enhancer is responsible for more than a half of the total promoter activity and is an essential element for the cell-specific activity of the Sertoli-specific region. In addition to the core enhancer, tandem Sp1 sites are also required for maximal activity of this region.

Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E₂). This increase in the inhibitory effects of E₂ is controlled by photoperiod and appears to be mediated, in part, by dopaminergic neurons in the retrochiasmatic area of the hypothalamus (A15 group). This study was designed to test the hypothesis that E₂ increases multiunit electrical activity (MUA) in the A15 during inhibitory long days. MUA was monitored in the retrochiasmatic area of 14 ovariectomized ewes from 4 h before to 24 h after insertion of an E₂-containing implant subcutaneously. In six of these ewes, MUA activity was also monitored before and after insertion of blank implants. Three of the 14 ewes were excluded from analysis because E₂ failed to inhibit LH. When MUA was recorded within the A15, E₂ produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E₂ did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E₂ increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E₂ negative feedback during long photoperiods.

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1β and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.

Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.

Testicular microvascular blood flow is known to exhibit vasomotion, which has been shown to be significantly altered in the short term following the repair of testicular torsion. This loss of vasomotion may ultimately be responsible for the loss of spermatogenesis observed after testicular torsion in rats. In the present study, testicular vasomotion and interstitial oxygen tensions were simultaneously measured prior to, during, and at various time points after repair of testicular torsion in the rat. Testicular torsion was induced by a 720° rotation of the testis for 1 h. Laser-Doppler flowmetry and an oxygen electrode were used to simultaneously measure vasomotion and interstitial oxygen tensions (PO₂), respectively. Pretorsion control testes had a mean blood flow of 16.3 ± 1.3 perfusion units (PU) and displayed vasomotion with a cycle frequency of 12 ± 0.2 cycles per minute and a mean amplitude of 4.2 ± 0.3 PU. Mean testicular interstitial PO₂ was 12.5 ± 2.6 mm Hg, which displayed a cyclical variation of 11.9 ± 0.4 cycles per minute with a mean amplitude of 2.8 ± 0.8 mm Hg. During the torsion period, both mean blood flow and interstitial PO₂ decreased to approximately zero. Upon detorsion, mean microvascular blood flow and mean interstitial PO₂ values returned to values that were not significantly different from pretorsion values within 30 min; however, vasomotion and PO₂ cycling did not return, even after 24 h. It was 7 days after the repair of torsion before a regular pattern of vasomotion and PO₂ cycling returned. These results demonstrate for the first time a correlation between testicular vasomotion and interstitial PO₂ cycling, and this correlation persists after the repair of testicular torsion.

The large increase in placental surface area and fetal villous vascular development in the third trimester of pregnancy requires degradation and reformation of the placental basal lamina. Degradation is carried out by matrix metalloproteinases (MMPs) secreted by adjacent cells. Although the gelatinases, MMP-2 and MMP-9, which are released by extravillous cytotrophoblasts (CTs) are believed to play crucial roles in early placental expansion, neither has been reported in third trimester villous trophoblasts nor has appropriate (basolateral) release of any MMP by the highly polarized syncytiotrophoblast (ST) been demonstrated. We demonstrated villous trophoblast expression of both MMP-2 and MMP-9 by in situ immunohistochemistry and by Western blot analysis and zymography of lysates and culture supernatants of highly purified villous CTs. We also found that epidermal growth factor (EGF)-stimulated CT differentiation into ST and stimulation by the phorbol diester, PMA, both increase MMP-9 secretion. The direction of MMP release was determined with confluent cultures of ST on porous membranes. We found that >90% of MMP-2 and MMP-9 were released from the basolateral surface. We conclude that villous STs express and release gelatinases from their basolateral surfaces in a regulated manner and suggest that such polarized release may be important to villous tissue remodeling.

Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25–33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.

Cimetidine (Tagamet) is a potent histaminic H2-receptor antagonist, extensively prescribed for ulcers and now available without prescription. Cimetidine is a known testicular toxicant, but its mechanism of action remains uncertain. Rats were treated i.p. with cimetidine either at 50 mg/kg or 250 mg/kg body weight for 59 days. Accessory sex organ weights, but not testis weight, were significantly reduced in the high dose treated groups. FSH levels were significantly elevated in both treated groups, but testosterone levels were unchanged. A high degree of variability characterized testis histology, with most tubules appearing normal and some tubules (15–17%) partially lacking or devoid of germ cells. Morphometry showed that although seminiferous tubule volume was not significantly changed, the volume of peritubular tissue was reduced in the high dose group. There was extensive duplication of the basal lamina, lamina densa in both apparently normal spermatogenic tubules and severely damaged tubules. Apoptotic peritubular myoid cells were also found. TUNEL labeling confirmed extensive apoptotic cell death in peritubular cells, but revealed apoptosis of vascular smooth muscle. Given that 1) peritubular myoid cell apoptosis occurs in apparently normal tubules, that 2) basal lamina disorders are found, and that 3) peritubular cells are lost from the testis, it is suggested that the primary event in cimetidine-related damage is targeted to testicular smooth muscle cells. This is the first in vivo-administered toxicant to be described that targets myoid cells, resulting in abnormal spermatogenesis.

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death.

Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4’-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy.

IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% ( IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 ± 2.9% (–IGF-I) to 8.7 ± 1.4% ( IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 ± 4.6 with IGF-I; 54.5 ± 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(−/−), iNOS(−/−), and nNOS(−/−) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.

Even though the epididymis produces an environment promoting sperm maturation and viability, some sperm do not survive transit through the epididymal tubule. Mechanisms that segregate the epididymal epithelium and/or the viable sperm population from degenerating spermatozoa are poorly understood. We report here the identification and characterization of HEP64, a 64-kDa glycoprotein secreted by principal cells of the corpus and proximal cauda epididymidis of the hamster that specifically binds to and coats dead/dying spermatozoa. The HEP64 monomer contains ∼12 kDa carbohydrate and, following chemical deglycosylation, migrates as a ∼52-kDa polypeptide. Both soluble (luminal fluid) and sperm-associated HEP64 are assembled into disulfide-linked high molecular weight oligomers that migrate as a doublet band of 260/280 kDa by nonreducing SDS-PAGE. In the epididymal lumen, HEP64 is concentrated into focal accumulations containing aggregates of structurally abnormal or degenerating spermatozoa, and examination of sperm suspensions reveals that HEP64 forms a shroudlike coating surrounding abnormal spermatozoa. The HEP64 glycoprotein firmly binds degenerating spermatozoa and is not released by either nonionic detergent or high salt extraction. Electron microscopic immunocytochemistry demonstrates that HEP64 localized to an amorphous coating surrounding the abnormal spermatozoa. The potential mechanisms by which this epididymal secretory protein binds dead spermatozoa as well as its possible functions in the sperm storage function of the cauda epididymidis are discussed.

Morphological changes in zona pellucidae (ZP) isolated from in vitro-matured (IVM) and ovulated porcine oocytes were compared before or after fertilization in vitro and in vivo, respectively, by using scanning electron microscopy (SEM). The ZP of some ovulated or IVM oocytes and in vivo- or in vitro-fertilized (IVF) zygotes were equally split into two halves while immersed in an enzyme-inhibitor solution, using a surgical blade. After washing, intact and ZP halves were fixed in 1% glutaraldehyde solution in 0.1 M cacodylate buffer, processed, and examined using SEM. The outer surface of ZP in ovulated oocytes had a mesh-like structure. The outer morphology in IVM oocytes was more smooth although the mesh-like structure was still visible at high magnification. In in vivo zygotes and IVM-IVF zygotes, this lysed, mesh-like structure was more obvious. The inner surface of ZP had some small depressions (orifices). The mean number of orifices per 100 μm² of ZP surface was larger in IVM oocytes as compared to ovulated ones. The number of orifices per 100 μm² decreased in IVM-IVF zygotes as compared to IVM oocytes; whereas, in vivo zygotes did not differ from ovulated oocytes. The mean diameter of intact ZP as well as their mean thickness was greater in ovulated oocytes than IVM oocytes. The mean thickness of the ZP was larger in ovulated oocytes than IVM ones. The ZP thickness was larger in zygotes than in in vivo oocytes, whereas that of IVM-IVF zygotes did not differ from that of IVM oocytes. These results indicate that the morphology of ZP and the ZP reaction at sperm penetration appears to be much different between IVM oocytes and ovulated ones.

In the present study we have shown that the genetic expression of prostaglandin (PG)F_2α receptor (R) and cyclooxygenase (COX)-2 increases in laboring rat myometrium. This finding was associated with a relatively weak contractile in vitro response (E_max) of isolated uterine strips when challenged with PGF_2α. Five days postpartum PGF_2α-R mRNA values exceeded those during labor while COX-2 mRNA was reduced to preparturient values. Maximal contractility of isolated strips stimulated with PGF_2α at this time was enhanced and EC₅₀ decreased. Oxytocin treatment of estrogen-primed nonpregnant rats down-regulated uterine contractile responsiveness to PGF_2α, leaving mRNA values for this receptor unchanged, whereas oxytocin receptor blockade with atosiban (an oxytocin receptor antagonist) left E_max unaltered. In contrast, atosiban treatment of pregnant rats resulted in a 2.5-fold increase in E_max and a considerably reduced EC₅₀ during labor when compared to untreated delivering rats. The increased contractile ability was associated with a threefold increase in PGF_2α-R mRNA production, indicating that the regulation by atosiban of the PGF_2α-induced response is exerted at the genetic level. Based on the present data we suggest that 1) PGF_2α-R stimulation may not primarily exert a contracting role in the normally delivering myometrium, and 2) the presence of the PGF_2α-R system in rat myometrium may explain the apparent functional redundancy of the oxytocinergic system during the process of birth in animals lacking oxytocin or where the oxytocin receptor is blocked. In this context PGF_2α receptor stimulation may, in the absence of oxytocin receptor stimulation, exert the contractile forces needed for proper propulsion of the fetus.

The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4°C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classes and only small changes were observed in phospholipid classes of seminal plasma. The motility, viability, and morphological integrity of spermatozoa decreased during storage. These changes in phospholipid content may be explained by membrane phospholipid lysis followed by endogenous metabolism or by a complex combination of lysis, metabolism, and peroxidation. They are likely to affect semen quality and the success of in vitro storage severely.

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.

The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis. Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules. Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II–III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-X_L was the prominent mRNA species. Caspases 1, 2, 3, and Bax mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-X_L and Bcl-X_S mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bax as well as Fas-FasL interactions.

In this study, the intracellular signaling mechanisms through which TNFα increases LDH(A4) activity/expression in primary cultures of porcine testicular Sertoli cells were investigated. Studies were focused on sphingomyelin hydrolysis pathway. Treatment of [¹⁴C]serine-labeled cells with TNFα (15 ng/ml, 0.8 nM) resulted in a transient decrease (∼20%) in cellular [¹⁴C]sphingomyelin and in an increase (∼27%) in [¹⁴C]sphingosine that remained elevated for at least 75 min. In the same experiments, no significant changes were detected in ceramide levels. Exogenous sphingosine stimulated LDH(A4) activity and LDHA expression in a dose-dependent manner (ED₅₀ = 8 μM of sphingosine). Such an increase in LDHA messenger RNA levels and LDH(A4) activity was detected at 24 h and was maximal after 48 h of treatment. Kinetically, the increase in LDH(A4) activity was similar whether Sertoli cells were treated with sphingosine (12 μM) or with TNFα (20 ng/ml). Although sphingosine mimicked the action of TNFα on Sertoli cells LDH(A4) activity and expression, the maximal stimulatory effect represented about 30% of TNFα maximal activity. Sphingomyelinase, C2 ceramide, sphingosine 1-phosphate, N,N-dimethylsphingosine, and phosphorylcholine had no significant effect on LDHA expression/LDH(A4) activity. Exogenous C2 ceramide increased LDH(A4) activity only in cytokine-treated cells, suggesting its involvement as sphingosine precursor in TNFα-stimulated LDH(A4) activity via the sphingomyelin hydrolysis pathway. The LDH(A4) activity stimulated by TNFα was decreased by 36.2% by an inhibitor of sphingosine formation, NH4Cl (4 mM), supporting a role of sphingosine in the TNFα effect. Moreover, bisindolylmaleimide (100 nM), a protein kinase C (PKC) inhibitor decreased significantly by 28.7% the TNFα effect on LDH(A4) activity but had no effect on the stimulating action of sphingosine, suggesting that if PKC is involved in TNFα action, the sphingosine effect on LDH(A4) is unrelated to the PKC activity or inhibition. Together, the present data suggest that in primary Sertoli cell cultures, TNFα stimulating action on LDHA expression is partly exerted via sphingomyelin hydrolysis pathway, sphingosine being the active metabolite.

Testicular steroidogenesis in the fetal rat is activated before the onset of pituitary gonadotropin secretion. We studied here whether the pituitary adenylate cyclase-activating polypeptide (PACAP) could regulate this early Leydig cell activity. Effects of the two PACAP forms, 27 and 38, were studied on cAMP and testosterone production of dispersed Leydig cells of embryonic Day (E) 18.5. Furthermore, PACAP and PACAP type I receptor mRNA expression were measured by reverse transcription-polymerase chain reaction (RT-PCR), and testicular PACAP concentations by RIA. The two peptides were highly potent stimulators of fetal testes. Doses as low as 10⁻¹⁸ mol/L of PACAP-27 and 10⁻¹⁷–10⁻¹⁶ mol/L of PACAP-38 significantly stimulated cAMP and testosterone production, with magnitude comparable to that evoked by hCG. These effects were specific for fetal Leydig cells, because PACAP-responsive control cells, including murine Sertoli and granulosa cell lines, only responded to concentrations ≥10⁻¹² mol/L. By RT-PCR, PACAP and its type I receptor mRNAs were expressed in fetal testis as early as E15.5. By Northern hybridization, PACAP mRNA was first detectable on Day 30 postpartum and increased thereafter. Both forms of PACAP peptides were clearly detectable in E17.5 testes, with decreasing levels thereafter. In conclusion, the steroidogenesis of fetal rat Leydig cells responds to very low concentrations of PACAP, which may be an important physiological regulator of this activity before the onset of pituitary LH secretion.

The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and Drosophila. Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.

Despite extensive research on the pathogenesis of polycystic ovary syndrome (PCOS), there is still disagreement on the underlying mechanisms. The rat model for experimentally induced polycystic ovaries (PCO)—produced by a single injection of estradiol valerate—has similarities with human PCOS, and both are associated with hyperactivity in the sympathetic nervous system. Nerve growth factor (NGF) is known to serve as a neurotrophin for both the sympathetic and the sensory nervous systems and to enhance the activity of catecholaminergic and possibly other neuron types. Electro-acupuncture (EA) is known to reduce hyperactivity in the sympathetic nervous system. For these reasons, the model was used in the present study to investigate the effects of EA (12 treatments, approximately 25 min each, over 30 days) by analyzing NGF in the central nervous system and the endocrine organs, including the ovaries. The main findings in the present study were first, that significantly higher concentrations of NGF were found in the ovaries and the adrenal glands in the rats in the PCO model than in the control rats that were only injected with the vehicle (oil or NaCl). Second, that repeated EA treatments in PCO rats resulted in concentrations of NGF in the ovaries that were significantly lower than those in non-EA-treated PCO rats but were within a normal range that did not differ from those in the untreated oil and NaCl control groups. The results in the present study provide support for the theory that EA inhibits hyperactivity in the sympathetic nervous system.

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F_2α analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 μg cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF_2α secretion. In experiment I, progesterone remained high and PGF_2α low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF_2α production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12,13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF_2α production only from Day 17 luteal cells. Thus, PGF_2α production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF_2α production is not explained by a difference in induction of PGHS-2 mRNA or protein.

The ovulatory process in mammals begins when an endogenous surge in LH circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.

Cocaine- and amphetamine-regulated transcript (CART) is a novel family of peptides, of which CART peptide fragments 55–102 and 62–102 are reported to be the endogenous, physiologically active peptides. Immunohistochemical studies with an antiserum directed against the CART peptide fragment 55–102 revealed CART-like immunoreactive (CART-LI) nerve fibers in the rat epididymis. The number was highest in the cauda epididymis and became progressively fewer toward the caput epididymis; the vas deferens exhibited an abundance of CART-LI fibers. Injection of the retrograde tracer Fluorogold (Fluorochrome, Inc., Englewood, CO) to the junction between the vas deferens and cauda epididymis labeled a large number of neurons in the major pelvic ganglion, some of which were CART-positive. Double-labeling the ganglion sections with tyrosine hydroxylase (TH) and CART antisera revealed that CART-LI and TH-LI were expressed in two distinct populations of ganglion cells. Some of the TH-LI cells in the ganglia, however, were covered with web-like CART-LI endings. The effects of CART peptide 55–102, referred to herein as CART, on anion secretion in the form of short circuit currents (Isc) were assessed in cultured epithelia. The CART (1 to 5 μM) applied to the basolateral or apical side of the cultured epithelia caused no significant responses on Isc, whereas lys-bradykinin (1 μM) produced a large Isc response in the same preparations. Our results show that CART-LI is present in a population of rat pelvic ganglion cells, which may give rise to CART-LI nerve fibers as observed in the vas deferens and the epididymis. The biological function of CART in the rat epididymis is not known, but it apparently is not involved in ion secretion across the epithelium.

Photoperiod is the major regulator of reproduction in temperate-zone mammals. Laboratory rats are generally considered to be nonphotoresponsive, but young male Fischer 344 (F344) rats have a uniquely robust response to short photoperiods of 8 h of light. Rats transferred at weaning from a photoperiod of 16 h to photoperiods of < 14 h of light slowed in both reproductive development and somatic growth rate. Those in photoperiods < 13 h of light underwent the strongest responses. The critical photoperiod of F344 rats can be defined as 13.5 h of light, but photoperiods of ≤ 12.5 h are required to fully suppress reproduction and somatic growth. This demonstrates that the 12-h photoperiod that is standard in some laboratory colonies would have significant effects on reproductive maturation and growth rate of this common rat strain. Young F344 rats in decreasing photoperiods that mimic natural change experienced delayed reproductive development and decreased growth rate to a greater extent and for a longer duration than those transferred at birth to a short photoperiod. The effects of gradual changes in photoperiod persisted for at least 12 wk after weaning. This indicates that young male F344 rats possess responses to photoperiod that would result in functional photoperiodism in a wild mammal.

Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.

Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of α-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed α-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.

Chronic aminonucleoside nephrosis in rats is an experimental analogue of human focal segmental glomerulosclerosis. This study was undertaken to define the effects of chronic nephrosis on the pituitary-ovarian axis and on fertility. Chronic nephrosis was induced by puromycin aminonucleoside and followed for 112 days. The estrous cycle was evaluated daily in all rats, whereas biochemical parameters, hormonal concentrations, and fertility were measured on Days 7, 14, 28, 56, 84, and 112 (n = 8). Animals were divided in four experimental groups: A, B, C, and D. Group A was used to determine LH, FSH, progesterone, and estradiol concentrations. Group B was used to evaluate fertility, and groups C and D were added to clarify the role of male rats in the fertility of nephrotic female rats. The results showed a persistent proteinuria in nephrotic rats; the estrous cycle of nephrotic animals was disrupted. The LH and estradiol concentrations were significantly low at all time points evaluated, whereas no significant changes were noted in FSH or progesterone values. In addition, fertility and litter size were diminished in nephrotic female rats. Interestingly, the presence of a male rat or its urine resulted in a positive influence on serum estradiol concentrations of nephrotic female rats. These data indicate that experimental chronic nephrosis results in a pituitary-ovarian dysfunction that is characterized by low LH concentration, hypoestrogenism, failure of the hormonal feedback control, and diminution of fertility. In addition, they show the positive effect of a male rat on the fertility of a nephrotic female, which strongly suggests the participation of pheromones.

We developed a novel promoter-based selection strategy that could be used to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.

We studied the effect of a mouse seminal vesicle autoantigen (SVA) on BSA-stimulated functions of mouse sperm. Uncapacitated, capacitated, and acrosome-reacted stages of sperm were morphologically scored, and the cellular zinc content was examined cytologically in a modified Tyrode solution at 37°C for 80 min. More than 85% of control cells remained uncapacitated. Addition of 0.3% SVA to the cell incubation did not affect the cell status. Approximately 65% of cells were capacitated in the incubation medium containing 0.3% BSA. Only 30% of the cells became capacitated after incubation with 0.3% BSA and 0.3% SVA together. The decapacitation effect by 0.3% SVA could be subdued by more than 3% BSA in the cell incubation. Whereas BSA did, SVA did not cause removal of Zn² from sperm, but SVA could suppress the BSA effect. The tyrosine phosphorylated proteins in sperm were detected after incubation in a modified HEPES medium containing 0.3% BSA and/or 0.3% SVA at 37°C for 90 min. Whereas BSA enhanced greatly, SVA did not cause phosphorylation of proteins in the range of M_r 40 000–120 000. The BSA-stimulated protein tyrosine phosphorylation could be suppressed by SVA in the cell incubation.

The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; α, β1, and β2), two novel PKC isoforms (ε and δ), and an atypical PKC isoform (ζ) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCβ1, PKCβ2, PKCδ, and PKCζ were translocated to the particulate fraction, and PKCε to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, Gö 6976, as well as the substantial depletion of PKCβ1 and PKCε and the partial depletion of PKCα and PKCδ by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCδ and PKCζ activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.