Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

Outer dense fiber proteins are dominant postobstruction autoantigens in adult Lewis rats.

Obstruction of the male reproductive tract commonly results in generation of antisperm autoantibodies. However, only a few of the sperm autoantigens recognized by these antibodies have been characterized. To identify postobstruction rat sperm autoantigens, sperm proteins were separated by two-dimensional(2-D) gel electrophoresis. Spots corresponding to proteins that were stained by at least 50% of postvasectomy rat sera on 2-D Western blots were removed from polyacrylamide gels and microsequenced by tandem mass spectrometry. From a total of 21 spots, 12 contained peptides that matched solely to either of two outer dense fiber proteins, odf1 or odf2. Six additional spots contained peptides comprising odf1 or odf2 and were accompanied by peptides representing other proteins. Only three spots lacked outer dense fiber peptides but did contain sequences of other known proteins. The results indicate that the outer dense fiber proteins odf1 and odf2 are dominant postobstruction autoantigens because they were detected in the majority of the immunoreactive protein spots examined. Possible explanations for this observation include the abundance of outer dense fiber proteins in spermatozoa, slow solubility, which may provide a sustained supply of antigen, and testis-specific expression during spermiogenesis.

Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath.

Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath. In many cases, DFS is familial, suggesting a genetic component. Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase. We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS. No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3. Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A. Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm. Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm. Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans.

Luteinizing hormone (LH) drives diverse intracellular calcium second messenger signals in isolated porcine ovarian thecal cells: preferential recruitment of intracellular Ca2+ oscillatory cells by higher concentrations of LH.

The present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 microg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85+/-5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1) [Ca2+]i oscillations with periodicities of 0.5 to 4.5 min(-1) (63+/-4.5%), 2) a [Ca2+]i spike followed by a sustained plateau (17+/-2.6%), 3) a [Ca2+]i spike only (5.8+/-2.6%); and 4) a [Ca2+]i plateau only (3.8+/-1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean [Ca2+]i interspike periodicity from 1.09+/-0.16 to 0.59+/-0.07 min(-1) (P < 0.05). Putative phospholipase C inhibition with U-73122 (10 microM) also abolished or frequency-damped LH-driven [Ca2+]i oscillations in 95+/-4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually [Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH's dose-responsive drive of [Ca2+]i oscillations in gonadal cells.

Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells.

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2- or no added Ca2+ with 100 microM EGTA or 100 microM CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67-fold, respectively over Ca2+-containing unstimulated control (P < or = 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 and 1.6 +/- 0.16-fold, respectively (P < or = 0.001 for FSH and P < or = 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450(scc) transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.49 and 2.9 +/- 0.45-fold, respectively (P < or = 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells declined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) promoter-luciferase reporter expression to 58 +/- 30% (and 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% (and 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 microM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450(scc) gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450(scc) promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

Influence of vitamin A injection before mating on oocyte development, follicular hormones, and ovulation in gilts fed high-energy diets.

Previous research revealed that treatment with vitamin A approximately 5 d before ovulation may increase litter size in weaned sows and improve embryonal survival in gilts fed high-energy diets that reduced embryonal survival. For the current study, the hypothesis was that administration of vitamin A before ovulation would alter development of follicles and oocytes in a way favorable to enhanced embryonal survival. (Landrace x Large White) x (Duroc x Hampshire) gilts (n = 44) were fed 11.0 Mcal ME x gilt(-1) x d(-1) beginning 7 d after second estrus and given (i.m.) corn oil or 1 x 10(6) IU of vitamin A (retinyl palmitate) on d 15 after second estrus. Gilts were checked for estrus every 4 h, mated naturally at third estrus, and assigned randomly to undergo midventral laparotomy beginning at 24 to 28, 28 to 32, 32 to 36, or 36 to 40 h after onset of third estrus. At laparotomy, ovulated oocytes and early-stage embryos were recovered from oviducts, and ovaries were removed for aspiration of oocytes and granulosa cells from unovulated follicles. Oocytes and embryos were stained for assessment of stage of development. Granulosa cells were cultured to assess their ability to secrete progesterone. Follicular fluid was assayed for progesterone, estradiol-17beta, IGF-I, and PGF2alpha. Treatment with vitamin A altered development of oocytes and embryos by decreasing the percentage at the germinal vesicle stage and increasing the percentage at advanced stages. Mean stage of development was increased by vitamin A, but variation in stage was decreased. Among follicles matched by meiotic stage of oocyte, follicular fluid concentrations of progesterone, IGF-I, and PGF2alpha were greater in vitamin A-treated gilts than in controls, but treatment with vitamin A in vivo did not affect LH-stimulated or unstimulated secretion of progesterone by granulosa cells in vitro. These data provide evidence that vitamin A may influence embryonic development by advancing resumption of meiosis and altering follicular hormonal environment during follicle maturation.

Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells.

Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca(2+)-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1-5 mm) and medium-sized (5-8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells. Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-beta1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-beta1 dose-response studies revealed an ED(50) of 0. 24-0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion. Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca(2+)-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 microM) stimulated intracellular free calcium ion concentrations ([Ca(2+)](i)) in single porcine theca cells. The [Ca(2+)](i) elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca(2+)](i) response. Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-beta and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.

Role of gonadotropin-releasing hormone pulse frequency in differential regulation of gonadotropins in the gilt.

We tested the hypothesis that different GnRH pulse frequencies will affect serum LH and FSH differently. Ovariectomized gilts (n = 6), immunized against GnRH, were given 200-ng pulses of GnRH agonist (GnRH-A) every 180 min for 3 days (pretreatment), followed by GnRH-A pulses every 30, 60, or 180 min for 3 days (treatment) in a Latin rectangle design. Mean gonadotropin concentrations did not change over time when GnRH pulses were administered every 180 min. Initiation of high GnRH-A pulse frequency (30 min) caused a robust increase in serum LH to 265% of the pretreatment level (p < or = 0.007) and a more moderate increase in serum FSH to 127% of pretreatment level (p < or = 0.02). After 66 h of frequent pulsing, desensitization had occurred and serum LH concentrations were similar to pretreatment concentrations, but serum FSH had decreased to 53% of pretreatment levels (p < or = 0.0008). After 72 h of treatment, 5 micrograms GnRH-A was infused to estimate residual releasable pools of LH and FSH, and the amounts of LH and FSH released were negatively correlated with GnRH-A pulse frequency. The results of this study imply that the LH surge is terminated because the pituitary gland becomes incapable of responding to an otherwise adequate stimulus, and not because of exhaustion of releasable LH pools. Our results confirm that in the pig the response to altered GnRH-A pulse frequency differs between LH and FSH. High GnRH pulse frequency is more effective in acutely releasing LH than FSH. Low pulse frequency of GnRH supports FSH synthesis and release, but is not as effective in increasing LH concentrations, while high GnRH pulse frequency inhibits FSH synthesis and release.