Proteins and Heparin Binding Proteins of Spermatozoa and Seminal Plasma in Beetal Bucks: Purification, Characterization and Role in In vivo Fertility

Background: The proteome analysis of seminal plasma and spermatozoa is of special relevance in livestock. Heparin binding proteins (HBPs) found in the seminal plasma of several mammals are shown to bind to sperm membrane and affect a series of events that contribute to normal fertility, such as sperm capacitation, formation of the oviduct reservoir and binding to the oocyte. Profiles of HBPs from seminal plasma and sperm membranes have been associated with sperm fertility. Although, HBPs present in the SP are described in several species, but little is known about HBPs in buck. Methods: Seminal plasma (SP) and sperm extracts (SE) of 13 bucks were subjected to heparin-sepharose affinity chromatography. Sperm extract, seminal plasma and purified HBPs and Non-HBPs were fractionated by SDS-PAGE. Total 78 females (6 per buck) were mated with 13 bucks. Bucks were divided into two groups, G-I (high fertile, 83.3-100% FR) and G-II (low fertile, 50-66.7% FR). Relationship between HBPs and fertility rate was observed. Result: SDS – PAGE of SP and SE resulted in resolution of 22 (10-240 kDa) and 21 (10-270 kDa) bands, respectively. Based on fertility rate 15 and 13 kDa proteins were absent in SP of higher number of GI-compared to G-II bucks. Fourteen bands ranging from 10 – 180 kDa and 10 – 150 kDa were separated from SP-NHBP and SP-HBP. SP-HBPs of 75, 35, 30, 28, 25 and 13 kDa were present in higher (28.6%, 42.5%, 26.2%, 40.5%, 14.3% and 36.2%) number of high fertile than low fertile bucks. NHBP and HBP purified from SE resolved into 11 bands ranging from 10 – 135 kDa and 10 – 120 kDa, respectively. SE-HBP of 53 kDa, 50/45 kDa and 25 kDa were present in higher percentage of high fertile than low fertile bucks.

Use of soluble sperm extract to improve cloning efficiency in zebrafish

During somatic cell nuclear transfer (SCNT), egg activation is required to initiate embryonic development. In zebrafish cloning, the reconstructed egg is activated by exposing it to hypotonic water. Egg activation using water-only is not capable of activating the same intracellular calcium release as fertilization which is required for proper embryonic development. Here we test whether the use of soluble sperm extract (SSE) can properly modulate the activation of reconstructed eggs during SCNT. We microinjected SSE from genomic-inactivated zebrafish sperm into unfertilized eggs and reconstructed eggs right after somatic cell nuclear transfer. We also evaluated the most effective approach for SSE microinjection. Microinjection of SSE (with 0.68 mg/ml of protein concentration) into non-activated eggs through the micropyle induced parthenogenetic development beyond the blastula stage, whereas all water-only activated eggs failed to enter the cleavage period. Microinjection of SSE at 1 mg/ml of protein concentration into non-activated reconstructed egg improved the developmental rate of cloned embryos in comparison to non-injected control clones. The cumulative survival time of cloned embryos injected with SSE was significantly longer than reconstructed eggs activated following sham injection (P<0.01). No significant difference was found among controls (P=0.32). SSE benefits both parthenogenesis and the survival cloned embryos which have never been reported in zebrafish. Further work is necessary to define the functional component(s) of SSE as well as the physiological pathway, to understand its principle of action and advance the utilization of SSE in cloning.

Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway

SummaryMatured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

33 PRODUCTION OF A CLONED FOAL USING MITOCHONDRIAL DNA-IDENTICAL OOCYTES

Recently we reported the birth of a viable foal produced by nuclear transfer (NT) using oocytes recovered from immature follicles of live mares by transvaginal ultrasound-guided aspiration (TVA; 2013 Theriogenology 79, 791–796). This procedure opens the door for production of mitochondrial DNA-identical cloned foals; typically, use of heteroplastic oocytes results in cloned offspring that have different mitochondrial DNA from that of the donor. We selected 2 mares (BL and SM) from the maternal line of the donor, a 23-year old stallion. Genetic analysis confirmed that the mares’ mitochondrial genotype was identical to that of the donor. Oocytes were obtained from the mares by TVA of all follicles ≥5 mm diameter, and were matured in vitro for 20 to 26 h. Donor fibroblasts were treated with 15 μM roscovitine for 24 h, then were directly injected into enucleated oocytes using a Piezo drill. Reconstructed oocytes were activated with 5 μM ionomycin for 4 min followed by injection with sperm extract, then incubation in 2 mM 6-dimethylaminopurine for 4 h. Oocytes from mare SM were assigned to treatment with either Scriptaid (500 nM) or Scriptaid plus vitamin C (50 μg mL–1) for 14 to 16 h, starting at the onset of 6-dimethylaminopurine exposure; mare BL did not provide sufficient oocytes for treatment grouping. Presumptive zygotes were cultured in vitro for 7 to 11 days and blastocysts were shipped for transfer to recipient mares, 1 embryo per mare. In mare BL, 10 aspiration sessions were conducted, 78 follicles were aspirated and 45 oocytes were collected, of which 4 were degenerating. After in vitro maturation, 12/40 (30%) oocytes were mature. Five of 12 oocytes lysed during manipulation; the remaining 7 were cultured and 1 blastocyst (14%) was obtained, which did not yield a pregnancy. In mare SM, 3 aspiration sessions were conducted and 53 oocytes were recovered from 81 follicles. After in vitro maturation, 31/53 (58%) were mature. Four oocytes were lysed during manipulation, 27 were cultured, and 4 blastocysts (15%) were produced, 2 from scriptaid treatment and 2 from scriptaid plus vitamin C. Transfer of these blastocysts yielded one pregnancy (scriptaid treatment); the mare delivered a healthy foal at 328 days of gestation. These results indicate that NT can be successful using low numbers of immature oocytes from selected mares. However, the individual mare may greatly affect the outcome in terms of oocyte number and quality; in this case, mare BL not only yielded fewer oocytes per aspiration session (4.5 v. 17.7 for mare SM; P < 0.001, t-test), but also fewer reconstructed oocytes per oocyte recovered (7/45 v. 27/53, respectively; P < 0.001, Fisher's exact test). Efficiency (14 to 15% blastocysts per reconstructed oocyte cultured; 1 foal from 5 embryos transferred) was similar to that achieved previously in our laboratory using heteroplastic oocytes. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, by Kit Knotts, and by Jack Waggoner.

Identification of phospholipase activity in Rhinella arenarum sperm extract capable of inducing oocyte activation

SummaryEgg activation, which includes cortical granule exocytosis, resumption and completion of meiosis and pronuclear formation culminates in the first mitotic cleavage. However, the mechanism through which the fertilizing sperm induces this phenomenon is still controversial. We investigated the effect of the microinjection of homologous sperm soluble fractions obtained by fast protein liquid chromatography (FPLC) from reacted sperm (without acrosome) and non-reacted sperm on the activation of Rhinella arenarum oocytes matured in vitro. The FPLC-purified sperm fraction obtained from reacted or non-reacted sperm is able to induce oocyte activation when it is microinjected. This fraction has a 24 kDa protein and showed phospholipase C (PLC) activity in vitro, which was inhibited by D-609 but not by n-butanol or neomycin, suggesting that it is a PLC that is specific for phosphatidylcholine (PC-PLC). The assays conducted using inhibitors of inositol triphosphate (IP3) and ryanodine receptors (RyRs) indicate that the fraction with biological activity would act mainly through the cADPr (cyclic ADP ribose) pathway. Moreover, protein kinase C (PKC) inhibition blocks the activation produced by the same fraction. Immunocytochemical studies indicate that this PC-PLC can be found throughout the sperm head.

RESPON PENGGUNAAN BERBAGAI BAHAN AKTIVATOR PADA AKTIVASI PARTENOGENESIS OOSIT KAMBING HASIL IVM

Parthenogenetic activation is one method that can be used to determine the quality of IVM oocytes results before further use to other reproductive technologies (IVF and transfer core). In parthenogenetic activation can be used various activators such as ethanol, Ca Ionophore, and Crude Sperm Extract (CSE). Therefore, the aim of this experiment is to know the response use a variety of materials activator of parthenogenetic activation of goat oocytes IVM.<br />The sample used in this experiment was oocytes aspirated from goat ovarian follicles taken from RPH Sukun of Malang. Oocytes were matured for 24 hr in TCM-199 supplemented with fetal bovine serum (FBS), follicle-stimulating hormone (FSH) and lutheinizing hormone (LH) at a temperature of 38,5oC and 5% CO2 in humidified air. After another 30 hours of in vitro maturation, they were then activated by various treatments. The treatment of experiment are treatment 1, activation using ethanol 7% for 7 minute, treament 2, activation using Ca Ionophore 20 µM for 7 minute. Treatment 3, activation using CSE 2,5 µg/ml for 2 hr.<br />Based on the result of research, it is showed that activation by using 7% ethanol for 7 minutes is able to produce cleavage rate of 70.40%. Activation by Ca Ionophore 20 μM for 7 minutes is able to produce cleavage rate of 52.75%. While the use of CSE activation with 2.5 ug / ml for 2 hours produces cleavage rate of 36.33%. Thus it can be concluded that the goat oocyte IVM able to respond to a variety of materials activators on parthenogenetic activation performed. The highest response given by the successive results goat IVM oocytes activated using 7% ethanol for 7 minutes, 20 μM Ca Ionophore for 7 minutes, and the CSE 2.5 microg / ml for 2 hours.<br /><br />Keywords: Parthenogenetic activation, goat oocytes IVM, etanol, calsium ionophore, Crude Sperm Extract (CSE). <br /><br />

Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P<0.05). In Experiment 3, blastocyst development rate was 6–11% after injection of sperm lyophilized from fresh or frozen–thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze–thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.