The requirement for oxidative metabolism of pyruvate during oogenesis in vivo was evaluated by inactivating Pdha1, a gene encoding an enzymatic subunit of pyruvate dehydrogenase complex, in murine oocytes at the beginning of the follicular growth phase. Immunohistochemical analysis revealed that Pdha1⁻ oocytes have dramatically reduced amounts of pyruvate dehydrogenase enzyme by the secondary follicle stage. Despite this reduction, these oocytes grow to normal size, are ovulated, and can be fertilized. Pdha1⁻ oocytes are, however, impaired in their ability to support embryonic development, as demonstrated by the failure of fertilized oocytes to develop beyond the one-cell zygote stage in vivo. Immunocytochemical evaluation showed that almost all (98.4%) ovulated Pdha1⁻ oocytes have not completed meiotic maturation and/or have gross abnormalities of the meiotic spindle and chromatin. Meiotic maturation is even more compromised when these oocytes are matured in vitro in the absence of cumulus cells or in the presence of the gap junction inhibitor 18-alpha glycyrrhetinic acid, indicating that cumulus cells can partially compensate for this enzymatic deficiency through a gap junction-mediated mechanism. Ovulated Pdha1⁻ oocytes were also shown to have reduced levels of total ATP content and NAD(P)H autofluorescence relative to oocytes without this enzymatic deficiency. These studies demonstrate that oxidative metabolism of pyruvate is essential for proper completion of oogenesis, serving as a vital source of energy during meiotic maturation. At earlier stages of oogenesis this metabolic pathway may not be necessary due to metabolic compensation by the granulosa cells.

The purpose of this study was to examine the effects of level of rumen inert fatty acids on developmental competence of oocytes in lactating dairy cows. Estrous cycles were synchronized in 22 cows on a silage-based diet supplemented with either low (200 g/day) or high (800 g/day) fat. A total of 1051 oocytes were collected by ultrasound-guided ovum pickup (OPU) in seven sessions/cow at 3–4 day intervals. Oocytes were matured, fertilized, and cultured to the blastocyst stage in vitro. Embryo quality was assessed by differential staining of Day 8 blastocysts. The high-fat diet reduced numbers of small and medium follicles. There was no effect on the quality of oocytes (grades 1–4) or cleavage rate. However, high fat significantly improved blastocyst production from matured (P < 0.005) and cleaved (P < 0.05) oocytes. Blastocysts from the high-fat group had significantly more total, inner cell mass and trophectoderm cells than the low-fat group (P < 0.05). Regression analysis showed negative effects of milk yield (P < 0.001), dry matter intake (P < 0.001), metabolizable energy intake (P < 0.005), and starch intake (P < 0.001) on blastocyst production in the low-fat group but not in the high-fat group. Within the low-fat group, blastocyst production was negatively related to growth hormone (P < 0.05) and positively related to leptin (P < 0.05). The low-fat group had higher nonesterified fatty acids than the high-fat group (P < 0.05). In conclusion, higher milk yields were associated with reduced developmental potential of oocytes in cows given a low-fat diet. Provision of a high-fat diet buffered oocytes against these effects, resulting in significantly improved developmental potential.

Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1–5 mm) and large (8–22 mm) bovine follicles and were treated with IGF2 for 1–2 days in serum-free medium, and steroid production, cell proliferation, specific ¹²⁵I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED₅₀) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED₅₀ of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and ³H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for ¹²⁵I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific ¹²⁵I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific ¹²⁵I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R.

Luteal inadequacy is a major cause of infertility in a number of species. During the early luteal phase, progesterone production requires the rapid growth of the corpus luteum (CL), which is in turn dependent on angiogenesis. In the present study, we examined the temporal changes in vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) during the follicular-luteal transition and CL development in the cow. Luteal VEGFA concentrations increased as the CL developed but were lower in the regressing CL. Conversely, luteal FGF2 concentrations were highest immediately postovulation in the collapsed follicle and declined as the CL developed. Furthermore, three FGF2 isoforms were present in the collapsed follicle, but only one isoform was detected in older CL. Interestingly, FGF2 concentrations increased in the regressing CL. Western blot analysis for SPARC showed the presence of two isoforms, which were constitutively expressed throughout CL development. Further studies investigated the regulation of FGF2 by LH, which showed that FGF2 concentrations in preovulatory follicular fluid were higher in those animals that had experienced an LH surge. Moreover, LH stimulated FGF2 production in dispersed luteal cells. Conversely, the LH surge had no effect on follicular fluid VEGFA concentrations. In conclusion, FGF2 was more dynamic than VEGFA and SPARC during the follicular-luteal transition, which suggests that FGF2 plays a key role in the initiation of angiogenesis at this time. Furthermore, it is likely that this is stimulated by the LH surge. The results also suggest that VEGFA and SPARC have a more constitutive, but essential, role in the development of the CL vasculature.

Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.

Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that has been implicated in cardiovascular disease. The objective of the present study was to determine the vasoactive effects and underlying mechanisms of S1P on adult human maternal arteries. The isometric tensions of the omental and myometrial arteries isolated from normal pregnant women at term were assessed in response to incremental doses of S1P in the presence or absence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The putative involvement of Rho-associated kinases (ROCKs) in intact arteries and in those permeabilized with α-toxin, to study agonist-dependent calcium-sensitization, was assessed with the inhibitor Y27632. Real-time RT-PCR established the presence of mRNA encoding the S1P receptors (S1P₁ to ₃), previously known as endothelial differentiation gene receptors (EDG1, 3 and 5), in both artery types. S1P induced a dose-dependent increase in the isometric tension of all the arteries. Y27632 reduced constriction due to S1P in intact arteries and reduced S1P-induced sensitization of contraction to submaximal activating Ca² in permeabilized arteries. L-NAME also modulated S1P vasoactive responses in a tissue-specific manner. Two subgroups of omental arteries were identified, one of which utilizes the NO pathway. In myometrial arteries, S1P evoked oscillatory constrictions, whereas pretreatment with L-NAME resulted in only tonic constrictions of unaltered peak magnitude. The prominent vasoactive actions of S1P in the maternal arteries of pregnant women are modulated by inhibitors of ROCKs and NO bioavailability. The subtle tissue-specific functional differences in the modulation of S1P actions by NO have important implications for vascular tone regulation by this bioactive circulatory metabolite during pregnancy.

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0–100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1–2 h of exposure to 50 mM alcohol. Exposure to 25–50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.

Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)^−/− mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope (TEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymidis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopc^−/− spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43°C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction—with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.

The male effect is a well-known phenomenon in female sheep and goats whereby a pheromone-induced activation of reproductive function occurs. However, the molecule(s) involved in this phenomenon are unknown. We investigated gene expression profiles for the induction of male effect pheromone synthesis using a PCR-based cDNA subtraction strategy. We constructed two subtracted cDNA libraries using mRNA from the skin of the head or rump region of orchidectomized male goats with or without pheromone induction using testosterone or dihydrotestosterone (DHT). Both libraries were assumed to contain genes whose expression increases with pheromone induction. Clones (n = 480) from each library were sequenced and identified using BLAST to reveal 115 and 239 types of sequences in the libraries of the head and rump region, respectively. Among these, 12 genes were expressed in both libraries. We conducted real-time PCR to further analyze their expression using cDNA samples derived from pheromone-producing or nonproducing skin from the head of an ovariectomized female goat with or without DHT implantation, respectively. For nine genes, we observed significantly increased expression in samples following DHT implantation. Among these, stearoyl-CoA desaturase 1 (SCD1) and elongation of long chain fatty acids family member 5 (ELOVL5) genes showed more than 100-fold higher expression levels in pheromone-positive samples, suggesting that the products of these genes may be important in pheromone synthesis.

Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an ∼60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.

A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.

Milk is used as a medium for sperm preservation. Caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm. Recently, we have shown that egg yolk, which is also widely used to preserve semen, protects sperm functions by preventing the binding to sperm of the major proteins of bull seminal plasma (BSP proteins), thereby preventing BSP protein-mediated stimulation of lipid loss from the sperm membrane. In the present study, we investigated whether milk caseins protect sperm in the same manner as egg yolk. Bovine ejaculates were diluted with skimmed milk permeate (skimmed milk devoid of caseins) or permeate that was supplemented with caseins and stored at 4°C for 4 h. In the semen diluted with permeate, sperm viability and motility decreased in a time-dependent manner. However, in semen diluted with milk or permeate supplemented with caseins, sperm functions were maintained. In addition, lower amounts of the BSP proteins were associated with sperm in semen diluted with milk or permeate supplemented with caseins, as compared to semen diluted with permeate. No milk proteins were detected in the sperm protein extracts. Furthermore, sperm diluted with milk or permeate supplemented with caseins showed 3-fold lower losses of cholesterol and choline phospholipids than sperm diluted with permeate during storage. Thus, milk caseins decreased the binding of BSP proteins to sperm and reduced sperm lipid loss, while maintaining sperm motility and viability during storage. These results support our view that milk caseins prevent the detrimental effects of BSP proteins on the sperm membrane during sperm preservation.

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

Blue crab vitellogenin (VTG) cDNA encodes a precursor that, together with two other Brachyuran VTGs, forms a distinctive cluster within a phylogenetic tree of crustacean VTGs. Using quantitative RT-PCR, we found that VTG was primarily expressed in the hepatopancreas of a vitellogenic female, with minor expression in the ovary. VTG expression in the hepatopancreas correlated with ovarian growth, with a remarkable 8000-fold increase in expression from stage 3 to 4 of ovarian development. In contrast, the VTG levels in the hepatopancreas and hemolymph decreased in stage 4. Western blot analysis and N-terminal sequencing revealed that vitellin is composed of three subunits of ∼78.5 kDa, 119.42 kDa, and 87.9 kDa. The processing pathway for VTG includes an initial hepatopancreatic cleavage of the primary precursor into ∼78.5-kDa and 207.3-kDa subunits, both of which are found in the hemolymph. A second cleavage in the ovary splits the ∼207.3-kDa subunit into ∼119.4-kDa and ∼87.9-kDa subunits. The hemolymph VTG profiles of mated and unmated females during ovarian development indicate that early vitellogenesis and ovarian development do not require mating, which may be essential for later stages, as VTG decreased to the basal level at stage 4 in the unmated group but remained high in the mated females. Our results encompass comprehensive overall temporal and spatial aspects of vitellogenesis, which may reflect the reproductive physiology of the female blue crab, e.g., single mating and anecdysis in adulthood.

Comprehensive understanding of the cellular mechanisms utilized by luteal cells in response to extracellular hormonal signals resulting in the normal synthesis and secretion of their steroid and peptide products has yet to be achieved. Previous studies have established that cAMP functions as a second messenger in mediating gonadotropin stimulated luteal progesterone secretion. Classically, increased intracellular concentrations of cAMP result in activation of protein kinase A (PKA), which in turn phosphorylates gene regulatory transcription factors. Recent studies demonstrate that non-PKA mediated actions of cAMP exist, yet the mechanisms are not well understood. In addition to gonadotropic hormones, such growth factors as insulin, insulin-like growth factor 1, and epidermal growth factor have been shown to modulate luteal steroid hormone synthesis and steroidogenic enzyme expression as either independent effects or via amplification or modulation of the action of gonadotropic hormones or cAMP. Thus, mechanisms independent of cAMP and also downstream to cAMP that do not involve PKA are likely to be important in steroidogenesis in mammalian cells. The present studies were performed to help define the cellular mediators involved in cAMP-stimulated progesterone expression. Our data demonstrate that, in an in vitro steroidogenic cell model, 1) cAMP-stimulated progesterone occurs in a manner that is independent of PKA, 2) neither phosphatidylinositol-3-kinase nor mitogen-activated protein kinase are involved in PKA-independent cAMP-stimulated progesterone production, 3) tyrosine kinase activity does mediate cAMP-stimulated progesterone production, and 4) cAMP directly activates the Ras protein. These data suggest novel mediators of cAMP-stimulated progesterone production.

To study the role of endothelial factors in luteal function, the dynamic profiles of genes for endothelin 1 (EDN1), its receptor subtypes, EDNRA and EDNRB, and angiotensin converting enzyme (ACE) were examined in corpora lutea (CL) obtained from rabbits on Days 4 and 9 of pseudopregnancy after prostaglandin (PG) F2α analogue (alfaprostol) treatment. The cell type distribution of EDN1 in the ovaries and its mechanisms of actions in vitro and in vivo were also studied. Positive immunostaining for EDN1 was localized in the luteal and endothelial cells, in granulosa cells of the follicles, and in the ovarian epithelium. The basal mRNA levels for EDNRA, EDNRB, and ACE were lower (P ≤ 0.01) in Day-4 CL than in Day-9 CL, whereas those for EDN1 did not differ between these two time-points. On Day 4, the luteal EDN1, EDNRA, EDNRB, and ACE mRNA levels were similarly increased two-fold (P ≤ 0.01) 1.5 h after alfaprostol injection, and did not show further changes in the subsequent 24 h. On Day 9, alfaprostol challenge transiently up-regulated (P ≤ 0.01) the luteal ACE transcripts at 1.5 h, and those of EDN1 at 1.5 h and 3 h, whereas the EDNRA and EDNRB transcript levels remained unchanged during the course of luteal regression. EDN1 decreased (P ≤ 0.01) progesterone release and increased (P ≤ 0.01) PGF2α secretion and NOS activity via the PLC/PKC pathway in Day-9 CL, but not in Day-4 CL, cultured in vitro. EDN1-induced, but not alfaprostol-induced luteolysis, was blocked by cotreatment in vivo with the ACE antagonist captopril. These findings support the hypothesis that PGF2α regulates luteolysis through intraluteal activation of the renin-angiotensin/EDN1 systems in CL that have acquired luteolytic competence.

The epididymis has traditionally been divided into the caput, corpus, and cauda regions, which are further organized into intraregional segments. In the rat and mouse, these segments have high degrees of transcriptional differentiation, and what has traditionally been called the initial segment of the rat epididymis actually consists of three transcriptionally different intraregional segments. These segments are regulated by endocrine, lumicrine, and paracrine factors, whose relative importance remains a topic of investigation. In the present study, 15-day unilateral efferent duct ligation (EDL) was used to deprive ipsilateral rat epididymides of lumicrine regulation. Segments 1–4 of EDL epididymides and contralateral, sham-operated tissues were collected individually. Microarray analysis of gene expression was used to determine the effect of lumicrine factor deprivation on the transcriptome-wide gene expression of each segment studied. More than 11 000 genes were detected as being expressed in each of the four segments examined. More than 2000 genes responded significantly to EDL in segment 1, although this number of genes declined in each succeeding segment. Segments 1 and 2 of control tissues were the most different transcriptionally and the most affected by EDL. In the absence of lumicrine factors, the four segments regressed to a transcriptionally undifferentiated state, which was consistent with the less-differentiated histology seen after EDL. Interestingly, for an individual gene, lumicrine factor deprivation could stimulate expression in some segments and suppress expression in other segments. These results reveal a higher complexity to the regulation of rat epididymal segments than heretofore appreciated.

Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E₂), for pregnancy recognition. Furthermore, E₂ increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E₂, and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E₂, P4 and E₂ (PE), or P4 and ZK and E₂ (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E₂ and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E₂ from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C₁₈-C₂₂) and very long-chain (VLC) (C₂₄-C₃₂) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7–10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.