Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn^K44A) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.

Estrogen-bound estrogen receptors (ER) α and β classically activate gene expression after binding to the estrogen response element in the promoter regions of target genes. Estrogen also has rapid, nongenomic effects. It activates several membranous or cytoplasmic kinase cascades, including the phosphatidylinositol 3-phosphate (PI3K/Akt) cascade, a signaling pathway that plays a key role in cell survival and apoptosis. Normal human endometrium is exposed to variable levels of steroid hormones throughout the menstrual cycle. We hypothesized that Akt phosphorylation in human endometrium may vary with the menstrual cycle and in early pregnancy and that fluctuations in estrogen level may play a role in Akt activation in endometrial cells. We analyzed Akt phosphorylation using in vivo and in vitro techniques, including Western blot, immunohistochemistry, and immunocytochemistry. Estradiol significantly increased Akt phosphorylation in endometrial cells. Rapid stimulation of Akt activation in cultured stromal cells was observed. Akt phosphorylation by estradiol was inhibited by the PI3K inhibitor, wortmannin, but not by the ER antagonist, ICI 182 780. The maximal effect on Akt activity was observed following 5–15 min of estradiol treatment. Our results suggest that estradiol may directly affect PI3K-related signaling pathway by increasing the phosphorylation of Akt in endometrial cells. Thus, estradiol may exert part of its proliferative and antiapoptotic effects by a nongenomic manner through the Akt signaling pathway.

Cell fate determination between self-renewal or differentiation of spermatogonial stem cells (SSCs) in the testis is precisely regulated to maintain normal spermatogenesis. However, the mechanisms underlying the process remain elusive. To address the problem, we developed a model SSC culture system, first, by establishing techniques to obtain enriched populations of stem cells, and second, by establishing a serum-free culture medium. Flow cytometric cell sorting and the SSC transplantation assay demonstrated that Thy-1 is a unique surface marker of SSCs in neonatal, pup, and adult testes of the mouse. Although the surface phenotype of SSCs is major histocompatibility complex class I⁻ Thy-1 α6-integrin αv-integrin^−/dim throughout postnatal life, the most enriched population of SSCs was obtained from cryptorchid adult testes by cell-sorting techniques based on Thy-1 expression. This enriched population of SSCs was used to develop a culture system that consisted of serum-free defined medium and STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders, which routinely maintained stem cell activity for 1 wk. Combining the culture system and the transplantation assay provided a mechanism to study the effect of single growth factors. A negative effect was demonstrated for several concentrations of basic fibroblast growth factor and leukemia inhibitory factor, whereas glial cell line-derived neurotrophic factor and stem cell factor appeared to have a positive effect on stem cell maintenance. The stem cell enrichment strategies and the culture methods described provide a reproducible and powerful assay system to establish the effect of various environmental factors on SSC survival and replication in vitro.

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)β superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFβ1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0–160 μg/ml) decreased the mitogenic activity of oocytes but only by ∼45% at the maximum dose of mAb. Just 5 μg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFβ pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGFβ1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.

In the present study, we cloned full-length cDNAs for epidermal growth factor (EGF), EGF receptor (EGFR), and three truncated forms of EGFR (EGFR15, 12, and 8) from the zebrafish ovary. Zebrafish EGF was predominantly expressed in the ovary and testis, while EGFR and its truncated forms were highly expressed in all tissues examined except the liver. In the ovary, the expression of EGF seemed to be more abundant in the follicles of early stages, while EGFR had much higher expression levels at later stages. Interestingly, although EGF was expressed in both the follicle cells and oocytes, its expression level was significantly higher in the oocytes. However, the expression of EGFR was mainly restricted to the follicle cells with little expression in the oocytes. The unique spatial patterns of EGF and EGFR expression within the follicle suggest that EGF may serve as a messenger from the oocyte to signal the follicle cells. EGF strongly stimulated the expression of both activin βA and βB, while it suppressed basal and hCG-induced follistatin expression in cultured follicle cells. These results, together with the evidence that EGF was predominantly expressed in the oocytes whereas EGFR was expressed in the follicle cells, strongly suggest that EGF is likely a potential paracrine/juxtacrine factor from the oocytes to regulate the function of the follicle cells.

Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le^x)-containing glycan (Galβ4[Fucα3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888–1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le^x-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa₅₆₈)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC₅₀ ∼180 nM), which at saturation, reduced Alexa₅₆₈-ZP3 binding by ∼70%. A Lewis A (Le^a)-capped glycan (Galβ3[Fucα4]GlcNAc) was also a potent inhibitor (IC₅₀ ∼150–200 nM), but at saturation, it reduced Alexa₅₆₈-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcβ4 or Galβ4 residues did not compete; neither did sialyl-Le^x (Neu5Acα 3Galβ4[Fucα3]GlcNAc-Lewis X) nor sulfo-Le^x (3′-O-SO₃-Lewis X). However, at saturation, Galα3Galβ4GlcNAcβ3Galβ4Glc reduced Alexa₅₆₈-ZP3 binding by ∼70% but with moderate apparent affinity (IC₅₀ ∼3000 nM). Fluorescence microscopy revealed that Alexa₅₆₈-labeled Le^x-Lac-BSA, Le^a-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le^a-Lac did not inhibit binding of Alexa₅₆₈-Le^x-Lac-BSA, and Le^x-Lac did not inhibit binding of Alexa₅₆₈-Le^a-Lac-BSA. Finally, Le^x-Lac and Le^a-Lac had an additive inhibitory effect on Alexa₅₆₈-ZP3 binding. Thus, Le^x is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le^a binding defines a different but less-abundant class of sites.

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le^x; Galβ4[Fucα3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888–1895). This glycan is recognized by ∼70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le^a; Galβ3[Fucα4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770–777). Herein, we test the hypothesis that Le^x- and Le^a-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le^x-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le^a-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le^x-containing neoglycoproteins. In contrast, neoglycoproteins containing β4-lactosamine (Galβ4GlcNAc), Lewis B (Fucα2Galβ3[Fucα4]GlcNAc), and sialyl-Le^x glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le^x- and Le^a-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le^x- and Le^a-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G_i protein. Thus, Le^x-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le^a-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.

Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes.

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)α and, to a lesser extent, interleukin (IL)1β inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNFα and IL1β, as well as by a direct effect on infected cells.

Studies assessed, either directly or indirectly, the role of GnRH in leptin-mediated stimulation of LH release in cattle before and after sexual maturation. In experiment 1, the objectives were to determine whether leptin could acutely accelerate the frequency of LH pulses, and putatively GnRH pulses, in prepubertal heifers at different stages of development. In experiment 2, we determined directly whether acute, leptin-mediated increases in LH secretion in the fasted, mature female are accompanied by an increase in GnRH secretion. Ten-month-old prepubertal heifers (experiment 1) fed normal- (n = 5) and restricted-growth (n = 5) diets received three injections of saline or recombinant ovine leptin (oleptin; 0.2 μg/kg body weight, i.v.) at hourly intervals during 5-h experiments conducted every 5 wk until all normal-growth heifers were pubertal. Leptin increased mean concentrations of circulating LH regardless of diet, but pulse characteristics were not altered at any age. In experiment 2, ovariectomized, estradiol-implanted cows (n = 5) were fasted twice for 72 h and treated with either saline or oleptin i.v. (as in experiment 1) on Day 3 of each fast. Leptin increased plasma concentrations of LH and third ventricle cerebrospinal fluid concentrations of GnRH, and increased the amplitude of LH and the size of GnRH pulses, respectively, on Day 3 of fasting compared to saline. Overall, results indicate that leptin is unable to accelerate the pulse generator in heifers at any developmental stage. However, leptin-mediated augmentation of LH concentrations and pulse amplitude in the nutritionally stressed, mature female are associated with modifications in GnRH secretory dynamics.

Overexpression of growth hormone (GH) as well as GH-deficiency dramatically impairs reproductive function. Decreased reproductive function as a result of altered GH release is, at least partially, due to changes at the hypothalamic-pituitary level. We hypothesize that hypothalamic somatostatin (SOM), the inhibiting factor of GH release from the pituitary, may play a central role in the “crosstalk” between the somatotropic and gonadotropic axes. In the present study we investigated the possible effects of a centrally applied SOM analog on the LH surge and the concurrent activation of hypothalamic GnRH neurons in female rats. To this end, female rats were treated with estradiol 2 wk after ovariectomy and were given a single central injection with either the SOM analog, octreotide, or saline just prior to surge onset, after which hourly blood samples were taken to measure LH. Two weeks later, the experimental setup was randomly repeated to collect brains during the anticipated ascending phase of the LH surge. Vibratome sections were subsequently double-stained for GnRH and cFos peptide. Following octreotide treatment, LH surges were significantly attenuated compared to those in saline-treated control females. Also, octreotide treatment significantly decreased the activation of hypothalamic GnRH neurons. These results clearly demonstrate that SOM is able to inhibit LH release, at least in part by decreasing the activation of GnRH neurons. Based on these results, we hypothesize that hypothalamic SOM may be critically involved in the physiological regulation of the proestrus LH surge.

In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.

Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of LβT2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature αT3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

We examined DNA methylation throughout the endogenous murine testis-specific phosphoglycerate kinase (Pgk2) gene and in human PGK2 promoter/CAT reporter transgenes in mouse spermatogenic cells before, during, and following the period of active transcription of this gene. We observed the gradual development of a domain of demethylation beginning over the promoter and then expanding approximately 1 kilobase in each direction within the endogenous Pgk2 gene. This demethylation domain develops in the absence of DNA replication and precedes other molecular changes that potentiate tissue-specific activation of this gene. Studies with transgenes show that a signal residing in the Pgk2 core promoter directs this gene-, cell type-, and stage-specific demethylation process. These results are consistent with a model in which regulated, tissue- and gene-specific demethylation initiates a cascade of subsequent molecular events required for tissue-specific activation of transcription during spermatogenesis in vivo.

The present study examined the gonadotropin surge-inducing actions of estradiol (E₂), both alone and with progesterone (P₄), in middle-aged, early persistent-estrous (PE) female rats that had become PE within 35 days. In addition, we also assessed the effect of P₄ on the mating-induced gonadotropin surges in these acyclic animals. Early PE rats were ovariectomized and received E₂ implants (Day 0). On Day 4, an s.c. injection of P₄ (0.5 mg/ 100 g body weight) at 1200 h markedly increased plasma P₄ and elicited both LH and FSH surges, whereas vehicle-treated controls displayed no rise in P₄ or gonadotropins. This observation confirms that at middle age, female rats no longer respond to the positive-feedback stimulation of E₂ on gonadotropin surges whenever the estrous cyclicity ceases. As PE continued, such a surge-inducing action of E₂ plus P₄ became diminished after 75 days of PE and disappeared thereafter. When caged with males, vehicle-treated early PE rats display a mating-induced increase in P₄ from the adrenal along with small gonadotropin surges. The amplitude of these mating-induced gonadotropin surges was enhanced by supplementation with exogenous P₄ in early PE rats. Our findings indicate that during the early phase of PE, the surge-inducing action of E₂ and P₄ remains intact but deteriorates as PE continues. Thus, a deficiency in P₄ secretion during aging may contribute to the diminished gonadotropin surge response in the hypothalamic-pituitary axis and the subsequent cessation of estrous cyclicity.

Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome α subunits at different stages of oocyte and early embryo development. The 20S proteasome α subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.

An additional vitellogenin gene (MeVg2) that is structurally different from MeVg1 was cloned and characterized from the shrimp Metapenaeus ensis. The MeVg2 gene consists of fewer exons-introns and is most likely evolved from the MeVg1 gene. The cDNA for MeVg2 is 8.0 kilobases (kb) in size, and the deduced MeVg2 precursor shared an overall 54% amino sequence identity to the MeVg1 gene of the same shrimp. As compared to the MeVg1 precursor, MeVg2 precursor consists of more potential subunit cleavage sites, suggesting that the precursor may be processed into many smaller subunits. The MeVg2 is expressed only in the hepatopancreas, and the expression level of MeVg2 in adult female increases from the early vitellogenic stage, reaching a maximum at the middle vitellogenic stage, and remains high toward the end of vitellogenic cycle. In addition to the 8-kb mRNA, smaller transcripts of 1.5–2.5 kb for MeVg2 were identified, and the 8-kb transcript only constitutes less than 10% of the overall MeVg2-derived transcripts. To confirm the presence of the small transcripts, we screened the shrimp hepatopancreas cDNA library and isolated two smaller MeVg2-specific cDNA clones. These clones shared greater than 99% overall identity to the corresponding C-terminal region of the MeVg2 precursor, suggesting that an alternative expression/ splicing of the MeVg2 gene occurred. By immunohistochemical analysis, vitellin-immunopositive signals were localized in the lumen and extracellular fraction of the hepatopancreas. Amino acid sequence determination of the tissue protein and secreted protein from the hepatopancreas revealed that the 76-kDa vitellogenin subunit is most likely processed into smaller-sized subunits. Taken together, these results suggest that the hepatopancreas is an important organ for the synthesis of vitellogenin and may contribute to vitellogenesis by producing a large quantity of smaller MeVg2 subunit for ovarian uptake.

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) and KILLER are a death-inducing ligand and receptor pair that belong to the TNF and TNF-receptor superfamilies, respectively. To date, only one apoptosis-inducing TRAIL receptor (murine KILLER [MK]) has been identified in mice, and it is a homologue of human Death Receptor 5. Whereas the expression of other death receptors, such as Fas and TNF receptor 1 have been documented in mammalian preimplantation embryos, no evidence currently demonstrates either the presence or the function of TRAIL and its corresponding death receptor, MK. Using reverse transcription-polymerase chain reaction and confocal immunofluorescent microscopy, we found that both TRAIL and MK are expressed from the 1-cell through the blastocyst stage of murine preimplantation embryo development. These proteins are localized mainly at the cell surface from the 1-cell through the morula stage. At the blastocyst stage, both TRAIL and MK exhibit an apical staining pattern in the trophectoderm cells. Finally, using the TUNEL assay, we demonstrated that MK induces apoptosis in blastocysts sensitized to TRAIL via actinomycin D. Taken together, these data are the first to demonstrate the presence and function of TRAIL and MK, a death-inducing ligand and its receptor, in mammalian preimplantation embryos.

An arrest in laying associated with either a polyovarian follicle (POF) or a polycystic ovarian follicle (PCOF) syndrome has been reported in turkey hens photostimulated at an early age with a constant-light photoperiod. Hens expressing the POF or PCOF syndrome had stopped laying for several weeks, but the ovary contained an increased number of mature-size and larger follicles (POF hens), which were cystic (PCOF) in some of the hens. Hens with the POF or PCOF syndrome had plasma progesterone (P₄) concentrations that were relatively high and without surges. We hypothesized that high plasma P₄ concentrations may block ovulatory surges of LH but not the growth or maintenance of hierarchical follicles leading to development of the POF or PCOF syndrome in turkey hens. In the first six studies, hens were photostimulated with either a 14L:10D or a 24L:0D photoperiod and, after laying for 1–38 wk, were then injected daily for up to 14 days with P₄ (up to 1.50 mg kg⁻¹ day⁻¹) and necropsied. At all ages, the oviposition rate was reduced at a P₄ dosage of 0.17 mg kg⁻¹ day⁻¹. With dosages of 0.33 mg kg⁻¹ day⁻¹ or greater, however, ovipositions stopped in most hens within approximately 2 days. For hens laying for less than 15 wk, oviductal weight and number of hierarchical follicles of P₄-injected hens were not different from control vehicle-injected hens, but the numbers of mature, cystic, and atretic follicles were increased. For hens laying for 38 wk, when treated with P₄, oviductal weight and number of hierarchical follicles decreased, but number of atretic follicles increased. No effect of photoperiod was found on egg production, oviductal weight, or follicle number, and none of the hens developed POF or PCOF syndrome in these experiments. Two additional experiments were conducted with hens early in the reproductive period that had been photostimulated with 14L:10D or 24L:0D and injected with P₄ (0.33 mg kg⁻¹ day⁻¹) for 10 or 12 days but not necropsied until 3 wk after the last injection. Most of the hens photostimulated with the 24L:0D photoperiod and injected with P₄, and a few of the hens photostimulated with the 14L:10D photoperiod and injected with P₄, had developed the PCOF syndrome when necropsied. The hens with the PCOF syndrome had high levels of P₄ when necropsied. From these studies, we concluded that the PCOF syndrome can be induced early in the reproduction period by photostimulating turkey hens with a 24L: 0D photoperiod, injecting them for 10 to 12 days with P₄ at a dosage of 0.33 mg kg⁻¹ day⁻¹, and then waiting 3 wk for the PCOF syndrome to develop.

Remodeling of the extracellular matrix (ECM) occurs during antral follicle growth, and the plasminogen activators (PA) have been implicated in this process in rodents. In the present study, we measured the expression and secretion of PA and the PA inhibitor protease nexin-1 (SerpinE2) in antral and basal bovine granulosa cells from small (<6 mm), medium (6–8 mm), and large follicles (>8 mm) during 6 days of culture in serum-free medium. Casein zymography revealed that the cells secreted predominantly tissue-type PA (tPA) with urokinase (uPA) being associated mainly with cell lysates, and Western blot demonstrated that the cells secreted SerpinE2. Overall, secreted tPA activity was higher in cultures of cells from small follicles compared with large follicles, and secreted SerpinE2 levels were higher in cultures of cells from large follicles. In cultures of cells from small follicles, secreted tPA levels increased with time of culture for antral but not basal cells, and SerpinE2 levels increased with time for basal but not antral cells. In cultures of granulosa cells from large follicles, tPA activity increased significantly with time of culture, whereas SerpinE2 levels decreased. Cell-associated uPA activity decreased with time in cells from medium and large follicles. Reverse-transcription polymerase chain reaction and Northern blot analysis showed that SerpinE2 secretion was regulated largely at the transcriptional level, whereas tPA secretion was not. The data suggest stage-dependent regulation of granulosa cell PA and SerpinE2 production, consistent with a role in ECM remodeling during follicle growth.

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17α,20β-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15–18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.

Amino acids and polyamines are essential for placental and fetal growth, but little is known about their availability in the conceptus in response to maternal undernutrition. We hypothesized that maternal nutrient restriction reduces concentrations of amino acids and polyamines in the ovine conceptus. This hypothesis was tested in nutrient-restricted ewes between Days 28 and 78 (experiment 1) and between Days 28 and 135 (experiment 2) of gestation. In both experiments, ewes were assigned randomly on Day 28 of gestation to a control group fed 100% of National Research Council (NRC) nutrient requirements and to an nutrient-restricted group fed 50% of NRC requirements. Every 7 days beginning on Day 28 of gestation, ewes were weighed and rations adjusted for changes in body weight. On Day 78 of gestation, blood samples were obtained from the uterine artery and umbilical vein for analysis. In experiment 2, nutrient-restricted ewes on Day 78 of gestation either continued to be fed 50% of NRC requirements or were realimented to 100% of NRC requirements until Day 135. Fetal weight was reduced in nutrient-restricted ewes at both Day 78 (32%) and Day 135 (15%) compared with controls. Nutritional restriction markedly reduced (P < 0.05) concentrations of total α-amino acids (particularly serine, arginine-family amino acids, and branched-chain amino acids) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids at both mid and late gestation. Realimentation of nutrient-restricted ewes increased (P < 0.05) concentrations of total α-amino acids and polyamines in all the measured compartments and prevented intrauterine growth retardation. These novel findings demonstrate that 50% global nutrient restriction decreases concentrations of amino acids and polyamines in the ovine conceptus that could adversely impact key fetal functions. The results have important implications for understanding the mechanisms responsible for both intrauterine growth retardation and developmental origins of adult disease.

Variation in reproductive capacity is common across the lives of all animals. In vertebrates, hypothalamic neurons that secrete GnRH are a primary mediator of such reproductive plasticity. Since social interactions suppress gonadal maturity in the African cichlid fish, Astatotilapia (Haplochromis) burtoni, we investigated whether the electrical properties of GnRH neurons were also socially regulated. Adult A. burtoni males are either territorial (T) and reproductively active or nonterritorial (NT) and reproductively regressed, depending upon their social environment. We compared the basic electrical properties of hypothalamic GnRH neurons from T and NT males using whole-cell electrophysiology in vitro. GnRH neurons were spontaneously active and exhibited several different activity patterns. A small fraction of neurons exhibited episodic activity patterns, which have been described in GnRH neurons from mammals. The type of activity pattern and spontaneous firing rate did not vary with reproductive capacity; however, several basic electrical properties were different. Neurons from T males were larger than those from NT males and had higher membrane capacitance and lower input resistance. In neurons from NT males, action potential duration was significantly longer and after-hyperpolarization characteristics were diminished, which led to a tendency for neurons from NT males to fire less rapidly in response to current injection. We predict this could serve to decrease GnRH release in NT males. These data are the first electrophysiological characterization of hypothalamic GnRH neurons in a nonmammalian species and provide evidence for several changes in electrical properties with reproductive state.

Platelet activating factor (PAF) is known as an autocrine growth/survival factor in mammalian preimplantation embryos. This study investigated the expression of porcine PAF receptor (PAFr) mRNA and its role in porcine in vitro fertilized (IVF) or somatic cell nuclear transfer (SCNT) embryo development. The expression of PAFr mRNA in IVF or SCNT blastocysts was shown by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Semiquantitative RT-PCR and Southern blot analysis demonstrated that PAFr mRNA was expressed during preimplantation embryo development, it was highly expressed through the 2-cell to 8-cell embryo stage, and it decreased at the morula stage. PAFr mRNA expression was detected steadily in IVF embryos, whereas it was varied at the 2-cell, 4-cell, and blastocyst stages in SCNT embryos. To determine the role of PAF in IVF and SCNT embryo development, embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of PAF (0, 0.037, 0.37, 3.72, or 37.2 nM). The PAF supplement significantly increased the rate of blastocyst formation in SCNT embryos, but not in IVF embryos. The PAF supplement for the entire 168 h of culture showed significantly higher blastocyst formation in SCNT embryos. Upregulation of PAFr mRNA by PAF in SCNT embryos indicated that the embryotrophic effect of PAF was mediated through its functional receptors in SCNT embryos. In conclusion, the present study demonstrated that PAFr mRNA was expressed in porcine IVF and SCNT embryos, and that PAF supplement improved the developmental competence of SCNT embryos through its specific receptors.

Paraspeckle protein 1 (PSP1) in humans is a recently identified component protein of a novel nuclear body, paraspeckle. The protein has a DBHS (Drosophila behavior, human splicing) motif that is found in PSF and p54^nrb/NonO proteins. These DBHS-containing proteins have been reported to be involved in various nuclear events such as DNA replication, transcription, and mRNA processing. Here we show that mouse paraspeckle protein 1 (mPSP1; encoded by the Pspc1 gene) has two isoforms with different C-termini lengths. Abundant expression of the longer isoform (mPSP1-α) and the shorter one (mPSP1-β) were observed in testis and kidney, respectively. Transiently expressed mPSP1-α was localized in nuclei, but mPSP1-β was localized in both nuclei and cytoplasm. These observations suggest that alternative splicing regulates tissue distribution and subcellular localization. Like other DBHS-containing proteins, mPSP1 has RNA-binding activity. In mouse testis, mPSP1-α was found in the nuclear matrix fraction. Furthermore, by coimmunoprecipitation, we confirmed that mPSP1 interacts with other DBHS-containing proteins, PSF and p54^nrb/NonO. Therefore, we conclude that mPSP1 may regulate multiple phases of important nuclear events during spermatogenesis.

The control of gene expression in reproductive tissues involves a number of unique germ cell-specific transcription factors. One such factor, ALF (TFIIAτ), encodes a protein similar to the large subunit of general transcription factor TFIIA. To understand how this factor is regulated, we characterized transgenic mice that contain the ALF promoter linked to either β-galactosidase or green fluorescent protein (GFP) reporters. The results show that as little as 133 base pairs are sufficient to drive developmentally accurate and cell-specific expression. Transgene DNA was methylated and inactive in liver, but could be reactivated in vivo by system administration of 5-aza, 2′-deoxycytidine. Fluorescence-activated cell sorting allowed the identification of male germ cells that express the GFP transgene and provides a potential method to collect cells that might be under the control of a nonsomatic transcription system. Finally, we found that transcripts from the endogenous ALF gene and derived transgenes can also be detected in whole ovary and in germinal vesicle-stage oocytes of female mice. The ALF sequence falls into a class of germ cell promoters whose features include small size, high GC content, numerous CpG dinucleotides, and an apparent TATA-like element. Overall, the results define a unique core promoter that is active in both male and female reproductive tissues, and suggest mouse ALF may have a regulatory role in male and female gametogenic gene expression programs.

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.

An improved understanding of reproductive physiology in nondomestic bovids is necessary for the development of assisted reproductive technologies (ARTs) for use in the conservation of endangered bovids. In this study, epididymal spermatozoa were recovered from blesbok (Damaliscus dorcas phillipsi), African buffalo (Syncerus caffer), springbok (Antidorcas marsupialis), and black wildebeest (Connochaetes gnou) following organized culls in South Africa. Our objectives were 1) to characterize the quality of epididymal spermatozoa, 2) evaluate the effectiveness of a cryopreservation protocol, and 3) compare postthaw sperm longevity (motility, viability, and acrosomal integrity) and functionality in two culture media with two capacitation reagents (caffeine and heparin). Following recovery, spermatozoa were diluted in EQ extender, slow-cooled, and frozen in the presence of 5% glycerol. Thawed spermatozoa were separated on a Percoll gradient and diluted in fertilization media (SOF for fertilization [SOFfert]; 0.6% BSA, 0.0 mM glucose, 25.0 mM NaHCO₃) or modified SOFfert (1.2% BSA, 1.5 mM glucose, 37.0 mM NaHCO₃) and either heparin or caffeine, and incubated for 6 h. Spermatozoa from these species maintained an average of 64% initial motility after thawing. Incubation medium and capacitation reagent had species-specific effects on the motility, viability, and acrosomal integrity of spermatozoa, suggesting ART procedures need to be optimized for each species. Springbok spermatozoa were also shown to be competent for in vitro fertilization. Information from this study concerning sperm physiology in blesbok, African buffalo, springbok, and black wildebeest will be useful in the development of ART for the conservation of these and other species of bovids.

Loss of calmegin, a testis-specific putative chaperone protein of the endoplasmic reticulum, leads to male sterility because the sperm show defects in migration into the oviduct and do not bind to the zona pellucida. To clarify the mechanism of defective migration, XY ⟷ XY chimeras were produced by aggregating wild-type embryos with embryos of transgenic mice lacking functional calmegin genes and expressing enhanced green fluorescent protein (EGFP) in their acrosomes. Chimeric ejaculates contained wild-type, nonfluorescent sperm as well as sperm with EGFP-tagged acrosomes and the defective calmegin gene. Transgenic, wild-type, and chimeric males were mated to wild-type females; however, only wild-type sperm were ever found within the oviducts. Calmegin-knockout sperm, even when they were combined in chimeric ejaculates with wild-type sperm, remained outside of the uterotubal junction. These findings indicate that the presence of wild-type sperm cannot compensate for the inability of calmegin-knockout sperm to enter the oviduct and that successful ascent into the oviduct depends on the capabilities of individual sperm.

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight]⁻¹ day⁻¹) or in the diet (0, 500, or 1000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 μM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)⁻¹ day⁻¹. Dietary equol at 500 or 1000 ppm produced total serum equol concentrations of 5.9 and 8.1 μM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8–20 mg (kg body weight)⁻¹ day⁻¹ and 1000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor α when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.

In general, the fertilizing ability of cryopreserved mouse spermatozoa is less than that of fresh spermatozoa. This ability is especially low in C57BL/6, the main strain used for the production of transgenic mice. To solve this problem, the relationship between cell damage and fertilizing ability in cryopreserved mouse spermatozoa was examined in this study. Sperm motility analysis revealed no significant difference among the motilities of cryopreserved C57BL/6J, BALB/cA, and DBA/2N sperm (67.6%, 43.4%, and 60.0%, respectively) after thawing. However, the results of in vitro fertilization (IVF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed a strong correlation between the frequency of aberrant spermatozoa (FAS) and fertilization rates (FR; C57BL/6J: FAS, 83.7%; FR, 17.0%; BALB/cA: FAS, 67.2%; FR, 24.2%; and DBA/2N: FAS, 10.2%; FR, 93.6%), and damage to spermatozoa was localized particularly in the acrosome of the head and mitochondria.

Reproductive performance in female birds improves with age, and this is generally attributed to experiences obtained during breeding. In temperate-zone species, experience with photostimulation during the first breeding year may prime the hypothalamo-pituitary-gonadal axis to respond to photic cues more rapidly or robustly in subsequent years. To test this idea, we captured 32 photorefractory juvenile (hence naive to photostimulation) female European starlings (Sturnus vulgaris) and held half of them (naive group) on a photoperiod of 8L:16D for 32 wk and the other half (experienced group) on 8L:16D for 12 wk, 16L:8D for 12 wk, and then 8L:16D for 8 wk. When we subsequently transferred all birds to 16L:8D, the increase in body mass, which may presage egg laying in the wild, was more robust in experienced than in naive females. Experienced females also showed a more robust elevation in plasma concentrations of the yolk-precursor protein vitellogenin, although naive females showed an initial rapid but transient rise in vitellogenin that we attribute to their extended exposure to short-day photoperiods prior to photostimulation. Finally, the photo-induced increase in diameter of the largest ovarian follicle, in plasma concentrations of luteinizing hormone, and in the number of septo-preoptic fibers relative to the number of cell bodies immunoreactive to GnRH was greater in experienced than in naive females. Thus, prior experience with photostimulation enhances some initial phases of photo-induced reproductive development and may explain, in part, why reproductive performance improves with age in temperate-zone birds.

Caloric restriction and hormone treatment delay reproductive senescence in female mammals, but a natural model of decelerated reproductive aging does not presently exist. In addition to describing such a model, this study shows that an abiotic signal (photoperiod) can induce physiological changes that slow senescence. Relative to animals born in April, rodents born in September delay their first reproductive effort by up to 7 mo, at which age reduced fertility is expected. We tested the hypothesis that the shorter day lengths experienced by late-born Siberian hamsters ameliorate the reproductive decline associated with advancing age. Short-day females (10L:14D) achieved puberty at a much later age than long-day animals (14L:10D) and had twice as many ovarian primordial follicles. At 10 mo of age, 86% of females previously maintained in short day lengths produced litters, compared with 58% of their long day counterparts. Changes in pineal gland production of melatonin appear to mediate the effects of day length on reproductive aging; only 30% of pinealectomized females housed in short days produced litters. Exposure to short days induces substantial decreases in voluntary food intake and body mass, reduced ovarian estradiol secretion, and enhanced production of melatonin. One or more of these changes may account for the protective effect of short day lengths on female reproduction. In delaying reproductive senescence, the decrease in day length after the summer solstice is of presumed adaptive significance for offspring born late in the breeding season that first breed at an advanced chronological age.

Cellular prion protein (Prp^C) is a glycoprotein usually associated with membranes via its glycosylphosphatidylinositol (GPI) anchor. The trans-conformational form of this protein (Prp^SC) is the suggested agent responsible for transmissible neurodegenerative spongiform encephalopathies. This protein has been shown on sperm and in the reproductive fluids of males. Antibodies directed against the C-terminal sequence near the GPI-anchor site, an N-terminal sequence, and against the whole protein showed that the Prp isoforms were compartmentalized within the reproductive tract of the ram. Immunoblotting with the three antibodies showed that the complete protein and both N- and C-terminally truncated and glycosylated isoforms are present within cauda epididymal fluid and seminal plasma. Moreover, we demonstrate that in these fluids, the Prp^C isoforms are both in a soluble state as well as associated with small membranous vesicles (epididymosomes). We also report that only one major glycosylated 25 kDa C-terminally truncated Prp^C isoform is associated with sperm from the testis, cauda epididymis, and semen, and this form is also present in the sperm cytoplasmic droplets that are released during maturation. In sperm, this C-terminal truncated form was found to be associated with membrane lipid rafts present in the mature sperm, suggesting a role for it in the terminal stages of sperm maturation.

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.

Chromatin remodeling during spermiogenesis is characterized by a series of nuclear protein replacements. Histones are replaced by transition nuclear proteins, which are in turn replaced by protamines. The transition nuclear proteins, TP1 and TP2, and the protamines, PRM1 and PRM2, are the major nuclear proteins involved in this process. Biochemical studies of mice with null mutations in one of the Tnp genes showed that the absence of one TP led to an apparent elevation in the amount of the remaining TP in the testis. To investigate the mechanism of changes of protein levels and effects of one Tnp mutation on other nuclear proteins, we used immunohistochemistry techniques to determine the distribution of these nuclear proteins. In contrast to previous biochemical analyses, which indicated that nuclear protein replacement was sequential with little overlap between the protein types, we found considerable overlap in the nucleoprotein types during spermiogenesis. The TPs, which appear in the nucleus before histone displacement is complete, were shared among genetically inequivalent spermatids. The absence of one TP did not affect the time of appearance of the other TP or of the protamines, but it did affect the displacement of the other TP, leading to its abnormal retention in the nucleus. The elevated levels of the remaining TP in Tnp-mutant mice appeared to be a consequence of the prolonged retention, rather than increased synthesis. Thus the absence of one of the TPs did not significantly affect transcription or translation of the other basic proteins, but it did affect posttranslational events.

To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.

Little is known about the ovary during aging in long-lived fish with respect to follicular stages and de novo oogenesis. We examined two species of rockfish, Sebastes aleutianus (rougheye rockfish) and Sebastes alutus (Pacific ocean perch). Fish were sampled offshore of British Columbia, age was estimated by otolith annuli, and the ovaries were examined histologically. In S. aleutianus, age up to 80 yr did not markedly alter the frequency distribution of oocytes, follicles, or their total numbers. Similarly, in a larger sample of S. alutus, the abundance of oocytes and follicles showed little age trend up through 77 yr. However, fish older than 50 yr lacked the largest and smallest oocyte size classes (40–60, >80 μm) and the smallest follicle size class (200– 350 μm), which results from the later seasonal developmental state of these older fish. These data provide evidence that oogenesis continues at advanced ages in these two species, in contrast with long-held assumptions about mammals. These species represent an iteroparous extreme in the spectrum of life history strategies and merit investigation to determine the mechanisms for such an extended reproductive life span.