SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition - Pires - 2013 - Developmental Dynamics - Wiley Online LibraryDevelopmental DynamicsVolume 242, Issue 12 p. 1405-1426 Research Article Free Access SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition Eusebio S. Pires, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCallie Hlavin, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorEllen Macnamara, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorKhadijat Ishola-Gbenla, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorChrista Doerwaldt, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCatherine Chamberlain, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorKenneth Klotz, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorAustin K. Herr, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorAalok Khole, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorOlga Chertihin, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorEliza Curnow, Division of Reproductive and Developmental Sciences, Washington National Primate Research Center, University of Washington, Seattle, WashingtonSearch for more papers by this authorSandford H. Feldman, Center for Comparative Medicine, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorArabinda Mandal, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorJagathpala Shetty, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCharles Flickinger, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorJohn C. Herr, Corresponding Author Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaCorrespondence to: John C. Herr, Box 800732, School of Medicine, Charlottesville, VA 22908. E-mail: jch7k@virginia.eduSearch for more papers by this author Eusebio S. Pires, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCallie Hlavin, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorEllen Macnamara, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorKhadijat Ishola-Gbenla, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorChrista Doerwaldt, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCatherine Chamberlain, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorKenneth Klotz, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorAustin K. Herr, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorAalok Khole, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorOlga Chertihin, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorEliza Curnow, Division of Reproductive and Developmental Sciences, Washington National Primate Research Center, University of Washington, Seattle, WashingtonSearch for more papers by this authorSandford H. Feldman, Center for Comparative Medicine, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorArabinda Mandal, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorJagathpala Shetty, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorCharles Flickinger, Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaSearch for more papers by this authorJohn C. Herr, Corresponding Author Department of Cell Biology, Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, VirginiaCorrespondence to: John C. Herr, Box 800732, School of Medicine, Charlottesville, VA 22908. E-mail: jch7k@virginia.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Background: Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Results: Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. Conclusions: The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies. Developmental Dynamics, 242:1405–1426, 2013. © 2013 Wiley Periodicals, Inc. INTRODUCTION SAS1B (a.k.a. ovastacin) has been shown to be an active zinc metalloprotease (Quesada et al., 2004; Sachdev et al., 2012) and a variety of biochemical proofs in the murine model have been advanced to support the conclusion that SAS1B interacts during fertilization with an intra-acrosomal ligand SLLP1, including yeast two hybrid analyses, co-immunoprecipitation, anatomical colocalization, and protein interaction affinity calculations (Mandal et al., 2003; Herrero et al., 2005; Sachdev et al., 2012). Targeted deletion of SAS1B/ASTL in female mice showed significant reductions in fertility but not complete infertility (Sachdev et al., 2012; Burkart et al., 2012) and antibodies to either SAS1B or SLLP1 inhibit sperm binding and fusion when assayed by in vitro fertilization (Mandal et al., 2003; Herrero et al., 2005; Sachdev et al., 2012). Burkart et al. (2012) proposed ovastacin is released from cortical granules and functions in the block to polyspermy by cleaving ZP2 and promoting a zona pellucida structure refractory to sperm penetration. The conjecture that ovastacin/SAS1B is involved in blastocyst hatching has also been advanced (Quesada et al., 2004) based on the hatching function of related metalloproteases in crayfish (Geier and Zwilling, 1998), although SAS1B\'s virtual absence in mouse blastocysts at the message and protein levels led Sachdev et al. (2012) to conclude that a role for ovastacin in mammalian blastocyst hatching was unlikely. The SAS1B protein is encoded by the ASTL gene located at a 2q11.1 chromosomal locus in humans, on chromosome 2A in chimpanzees, chromosome 13 in rhesus, chromosome 17 in canines, chromosome 11 in cattle, chromosome 2F in mice, and chromosome 3q26 in rats. In humans ASTL is flanked at its 5′ end by the Dusp2 gene which encodes dual specificity phosphatase 2 and by Adra2b, coding for adrenergic receptor alpha 2b, at its 3 prime-end. This gene association is conserved from mouse to man. The human ASTL locus at 2q11.1 is perfectly syntenic to the loci in cattle and nonhuman primates as well as to mouse, rat and dog, although the order of the flanking genes is reversed in this latter group. The astl gene in mouse oocytes undergoes remarkable alternative splicing involving at least six distinct mRNAs, resulting in SAS1B protein isoforms ranging from 31 to 62 kDa in apparent mass (Sachdev et al., 2012). Although it is probable that selected SAS1B isoforms engage in different biological functions such as ZP2 proteolysis and sperm-oolemmal interactions, the assignment of different functions to various SAS1B splice variants is presently incomplete. Given the importance of fertilization for the continuity of species and evolutionary fitness, one might predict that reproductive proteins required for interaction between sperm and egg would be highly conserved. In general, the requirement of molecular recognition between interacting proteins should result in sequence conservation, and indeed, there is proteome-wide evidence that interaction interfaces are more highly conserved than other surface residues (Mintseris and Weng, 2005). It is likely that egg proteins involved in sperm-oocyte recognition respond to evolutionary pressures for protein conservation, that the interacting surfaces of proteins mediating sperm-oocyte interactions co-evolve, and that changes in one protein involve adaptive compensations to these changes in the other partner in the interacting pair. It is also possible that proteins involved in various steps of fertilization are tightly regulated with respect to their timing and duration of expression. Proteins that are unique to gametes may be given particular consideration as targets for contraceptive strategies. The mainstay of population control in domestic mammals has been surgical sterilization especially for cats and dogs (Cathey and Memon, 2010). This invasive technique involves surgical equipment, anesthesia, a skilled veterinarian, adequate funds and a recovery period from operative pain. Thus nonsurgical approaches to contraception in veterinary species and companion animals would provide an alternative for population control especially for stray animals, and particularly in developing countries. Vaccines, small molecule inhibitors of enzymes and other proteins, and the use of antibody-drug conjugates [ADCs] or peptide-drug conjugates may provide novel strategies for development of nonsteroidal contraceptives some of which might directly target the mammalian oocyte. Because contraceptives are used to treat otherwise healthy animals, side effects and off-target effects must be minimized. Safety and efficacy criteria to achieve selective and reversible contraceptive targeting include the identification of candidate molecules whose expression is confined to the gametes, selection of candidate molecules that are not expressed in normal non-reproductive tissues (Ada and Griffin, 1991). The present study was undertaken to examine the developmental ontogeny of SAS1B in neonatal, prepubertal, pubertal, and adult rat ovaries especially to determine the relationship of SAS1B expression to stages of folliculogenesis. A panel of nongonadal tissues and adult ovaries spanning several mammalian orders were studied to define the tissue distribution of SAS1B and to particularly to examine the relationship of SAS1B expression to stages of folliculogenesis in several eutherians. Immunoreagents to both recombinant mouse and recombinant human SAS1B were developed and patterns of immunohistochemical staining for SAS1B were compared. The common properties of SAS1B expression observed across a variety of eutheria in this study, including precise temporal translation of SAS1B protein in growing oocytes, tight developmental regulation at the primary-secondary follicle transition, absence of SAS1B in the adult ovarian reserve, and the appearance of SAS1B on the surface of ovulated oocytes, suggest very tight regulation of the astl gene across mammalia and highlight the astl gene product as a candidate contraceptive target. To produce recombinant human SAS1B a construct spanning amino acids 55–368 of hSAS1B, including domains of high inter-species conservation, was subcloned into the E. coli expression vector pET28 and the construct was transformed into the E. coli expression host BL21. hSAS1B was expressed with a six-histidine tag and purified by IMAC (Fig. 1A–D). The pET28/SAS1B-expressed, IMAC-purified fraction was further prep-cell purified and peak fractions eluted from the prep-cell were pooled, concentrated, electrophoresed by reducing SDS-PAGE, stained with silver, then electro-transferred, and immunostained with anti-His antibody. The purified recombinant SAS1B preparation showed a single band that was immunoreactive with the anti-His antibody (Fig. 1E–G) and migrated at the expected molecular weight of ∼35 kDa, demonstrating the high purity of the immunogen preparation. The silver stained sample was sent for protein sequencing by mass spectrometry and ∼49% of the recovered tryptic digested peptides matched to human ASTL, confirming the authenticity of the purified recombinant protein (Fig. 1H). Figure 1Open in figure viewerPowerPoint Cloning of human SAS1B and production of rabbit polyclonal antibodies. A 942 bp SAS1B expression construct was cloned (A) in the pET28b+ expression vector (B) including 6-His-tags at c-terminus resulting in a ∼35 kDa protein (C,D). Coomassie stained fractions revealed a protein band at 35 kDa (red arrow, C) in inclusion body (P, pellet) fraction after IPTG induction and not in supernatant fraction (S) which was confirmed by anti-his-tag (D). Protein was purified by nickel affinity chromatography followed by preparative cell electrophoresis. SAS1B fractions 18–22 (E) tested positive with anti-his-tag (F). The far left lane contains the positive control pellet of the IPTG induced fraction. Positive fractions were pooled, concentrated, and electrophoresed following silver staining revealing a single band at ∼35 kDa (G). Mass spectrometry confirmed SAS1B peptides in blue and red after tryptic digestion when blasted in NCBI databases (H). Purified SAS1B protein was injected in 2 male rabbits and titers of post immunized sera (RbIM) were compared with the preimmune sera (RbPIM) by ELISA (I). Rabbit #2 with slightly higher titers was selected for future bleeds and used for subsequent studies. The RbIM reacted to SAS1B protein, whereas the RbPIM from the rabbit did not in Western blot (J). Anti-hSAS1B Antibodies Recognize Recombinant and Native SAS1B Proteins on Western Blots and by IHC Following three immunizations with the purified 35 kDa recombinant human SAS1B protein, the resulting immune rabbit sera (Rb1/2 IM) were compared with preimmune (Rb1/2 PIM) sera from the identical animals by ELISA using recombinant human SAS1B as the target. Two rabbits strongly responded to recombinant SAS1B by ELISA with serum dilutions of 10−11 showing statistically significant binding above PIM. The sera from rabbit #2 showed highest titer (Fig. 1I). This serum immunoreacted to recombinant SAS1B on Western blot whereas the preimmune serum from this animal did not (Fig. 1J). Both rabbit sera were also tested for immunoreactivity to native SAS1B in mouse ovarian sections. A 100-fold diluted Rb2IM serum immunoreacted strongly and specifically with fixed and embedded oocytes of secondary follicles on ovarian sections (black arrowheads in Fig. 5Q) in comparison to Rb1IM serum which at a similar dilution gave very weak oocyte signals (data not shown). No immunoreactivity with Rb2IM serum was seen with oocytes resting within primordial and primary follicles (white arrowheads in Fig. 5Q). Thus, the Rb2IM serum generated to a recombinant human SAS1B immunogen identified epitopes on recombinant human SAS1B and cross reacted with native mouse SAS1B in fixed and processed mouse ovarian sections. This successful dual screening method on recombinant and native SAS1B targets represents a useful strategy to select immunoreagents suitable for immunocytochemistry that is applicable to future selection of monoclonal antibodies to the SAS1B target. The Rb2IM serum generated to the recombinant human SAS1B immunogen, a previously reported guinea pig antibody (GPgIM) generated to a recombinant mouse SAS1B immunogen (Sachdev et al., 2012), and a commercially available rabbit polyclonal antibody to the pro-peptide domain of human ASTL (Abcam, MA) were used in immunochemical studies below. A phylogenetic tree incorporating the SAS1B protein sequence was created using the ClustalW program showing the inferred evolutionary relationships among various eutherian species based upon similarities and differences in protein sequences compared with human SAS1B as provided by NCBI. The dendogram (Fig. 2A) revealed the closest SAS1B orthologues to humans are those of the macaques, followed by cattle, carnivores, and rodents. The Drosophila SAS1B is more distantly related to the human SAS1B molecule. The sequence data accompanying the dendogram (Fig. 2B) reveal the Astl gene and SAS1B protein homologies, respectively, compared with the human SAS1B sequence. Macaques show identities of 96.3 and 98.1%; cattle 73.6 and 80.1%; dogs 73.1 and 79.8%, rats 71.3 and 76.4%, and mice 68.7 and 75.2% at the protein and DNA levels, respectively. However, 40.5 and 48.7% identity between human and Drosophila SAS1B was noted at the level of protein and DNA, respectively. Sequences of SAS1B orthologues encoded by Astl from Mammalia including humans, macaque, rat, dog, pig, and sheep were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and were aligned by ClustalW. For optimal alignment, gaps (−) were introduced into the sequences. A consensus sequence was created using identities (*) in all six sequences (accession #: human, NP_001002036; macaque, XP_002799475; rat, NP_001099974; dog, XP_003639630; pig, XP_003481181; sheep, XP_004007400). All sequences except macaque and sheep revealed a predicted signal peptide (residues in italics) followed by a signal peptide cleavage site between ILG-AP (marked, ♦). All sequences revealed a putative transmembrane domain (residues in shade) using TopPred algorithm for eukaryotes (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred). In all species, SAS1B revealed a conserved zinc binding metalloprotease active site (marked in closed box, catalytic residue↓). The number of residues in each protein is shown at the end of each sequence (Fig. 2C). A stick diagram indicates the main domains and their respective amino acids positions (Fig. 2D) including a N-terminus signal peptide 1–23, pro-peptide 24–90, followed by a proteinase domain 91–279 (which houses the putative transmembrane domain 143–163 and the catalytic site 182–192 where zinc atoms are coordinated at positions 182, 186, 192), followed by a unique C-terminus extending from 280–431. Figure 2Open in figure viewerPowerPoint Phylogenetic display of sequence identity of SAS1B across several species. As predicted from evolutionary divergences the dendogram (A) and table (B) reveal the closest SAS1B orthologue to human is the nonhuman primate and farthest is Drosophila. For optimal alignment, gaps(−) were introduced into the sequences. A consensus sequence was created using identities(*) in all six mammalian sequences (accession no.: human NP_001002036; macaque XP_002799475; rat NP_001099974; dog XP_003639630; pig XP_003481181; sheep XP_004007400). All SAS1B sequences except macaque and sheep revealed a predicted signal-peptide (residues in italics) followed by signal-peptide cleavage site between ILG-AP (marked, ♦). Sequences revealed a putative transmembrane domain (residues in shade) using TopPred algorithm for eukaryotes (http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::toppred). Sequences revealed conserved zinc binding metalloprotease active site (marked in closed box, catalytic residue↓). Number of amino acid residues is shown at the end of sequence (C). A stick diagram indicates major SAS1B domains (D) and defines amino acid boundaries of signal-peptide 1–23, pro-peptide 24–90, proteinase domain 91–279 (which houses transmembrane domain 143–163 and catalytic domain 182–192 where zinc atoms are coordinated at positions 182, 186, 192) followed by a distinct c-terminus encompassing 280–431. The high SAS1B sequence conservation among different mammals (B) is notable and underlies cross reactivity of antisera to SAS1B observed in different species. Major Forms of SAS1B in Ovaries of Various Species are 44–46 kDa Proteins With Additional High Molecular Weight Variants Noted If SAS1B behaves like other zinc metalloproteinase members of the astacin family which are known to exhibit highly variable structural and functional features (Semenova and Rudenskaya, 2009), SAS1B is predicted to have a prodomain that is endoproteolytically cleaved to yield an active enzyme. The timing and locale of such processing in vivo is presently unknown. Ovarian tissue protein extracts from various eutherian species were analyzed by Western blot to identify and compare SAS1B protein isoforms using SAS1B polyclonal antibodies. Ovarian protein extracts (Fig. 3), displayed immunoreactive ∼45 and 52 kDa (predicted 47.5 kDa, 45.2 kDa, 41.4 kDa, 43.7 kDa, 42.4 kDa, 44.8 kDa) murine SAS1B protein bands (A1), ∼45 and 55 kDa forms (predicted 44.9 kDa) in rat (B1), a major ∼44 kDa form in cat (C1), ∼37, 46, 55, 75 kDa forms (predicted 56.3 kDa) in dog (D1), ∼46 kDa in pig (E1), ∼44 kDa (predicted 41.7 kDa) in sheep (F1), and a ∼46 kDa form (predicted 41 kDa) in the crab-eating macaque (G1). The PIM sera showed no immunoreactivity to any protein in the ovarian extracts (A2-G2). In sum, the major immunoreactive SAS1B proteins detected in ovarian protein extracts in all species migrated at 44–46 kDa, with some species showing additional 52–55 kDa SAS1B isoforms. Thus, in Western blot analyses predicted masses match closely to the experimentally obtained apparent masses using the antibodies generated for this study. The 44–46 kDa isoforms appear to be the major form of the protein in all these species although higher molecular weight proteins, possibly isoforms, do exist, perhaps including posttranslational modifications. Figure 3Open in figure viewerPowerPoint Western blots detecting SAS1B in Eutherian ovarian protein extracts. After separation on 10% SDS PAGE blots were incubated with GPgPIM or GPgIM sera for all samples except macaque, where RbPIM and Rb2IM sera were used. Blots were developed using ECL. Ovarian protein extracts displayed immunoreactive ∼45 and 52 kDa murine SAS1B protein bands (A1), ∼45 and 55 kDa forms in rat (B1), a major ∼44 kDa form in cat (C1), ∼37, 46, 55, 75 kDa forms in dog (D1), ∼46 kDa in pig (E1), ∼44 kDa in sheep (F1) and a ∼46 kDa form in the crab-eating macaque (G1). PIM sera showed no immunoreactivity to any protein in any ovarian extract (A2–G2). SAS1B Protein Restriction Within the Ooplasm Persists Irrespective of the Stage of the Estrus Cycle in Mice Murine ovaries which had not received exposure to exogenous hormones were harvested at proestrus (P), estrus (E), and metestrus (M; Fig. 4), as defined by vaginal cytology (P4, E4, M4, in Fig. 4) and examined for SAS1B immunoreactivity. Within the ovary SAS1B protein was expressed only in oocytes and no changes in the intensity of SAS1B staining within oocytes were noted at different stages of the estrus cycle. The ooplasm of only growing oocytes in the secondary ovarian follicles and subsequent stages showed immunoreactivity as indicated by the brown DAB precipitate of the HRP reaction (Fig. 4, P2, E2, M2, and higher magnifications as seen in Fig. 4, P3, E3, and M3). PIM sera showed no immunostaining to any cell types (Fig. 4, P1, E1, and M1). Figure 4Open in figure viewerPowerPoint SAS1B protein expression in ovaries during the estrus cycle. Mouse ovaries were harvested during pro-estrus (P), estrus (E), and metestrus (M) stages as defined by vaginal cytology (P4, E4, and M4, light microscopy images). Ovarian sections stained with GPgPIM serum showed no SAS1B immunoreactivity in any ovarian cell type at any stage of the estrus cycle as seen in series 1. GPgIM serum showed strong immunostaining to oocytes in developing follicles at all stages of mouse estrus cycle (series 2 in black arrowheads, red arrowhead demonstrates a transition follicle), detailed at higher magnification in series 3. Quiescent oocytes within primordial follicles and growing oocytes within primary follicles (as seen in white arrowheads) showed no immunoreactivity for SAS1B protein. Magnification: ×40 for panels in series 1 and 2; ×200 for series 3 and 4. Figure 5Open in figure viewerPowerPoint Survey of SAS1B protein translation by immunohistochemical localization in normal adult mouse tissues. SAS1B immunoreactivity using the Rb2IM serum was only observed within a single unfertilized ovulated oocyte resting within the uterine lumen (P) and in oocytes within the ovary (Q) in a survey of tissues including cerebral cortex (A), thyroid (B), thymus (C), heart (D), liver (E), skeletal muscle (F), lung (G), spleen (H), kidney (I), adrenal (J), pancreas (K), small intestine (L), testis (M), epididymis (N), oviduct (O), uterus (P), and ovary (Q). Other than oocytes within the uterus (black arrow) and ovary (black arrowhead), SAS1B immunoreactivity was observed in no other cell type in any normal adult tissue. PIM sera controls were unreactive with all tissues including oocytes within the uterine lumen (data not shown) and ovaries (R). Panel S shows oocyte-specific staining pattern (black arrowhead) obtained with a commercially available pro-peptide ASTL antibody, which is identical to that of the Rb2IM antibody, confirming the authenticity of immunoreagents developed in the laboratory. White arrowheads in panels Q and S show no staining in the oocytes of primary follicles with Rb2IM and pro-peptide antibody respectively. Magnification: ×200 for panels A–O, Q–S; ×400 for panel P. The distribution of SAS1B protein was probed using HRP immunohistochemistry in a panel of 12 somatic (panels A–L) and 5 reproductive (panels M–S) mouse tissues using the Rb2IM antibody. SAS1B protein expression was not detected in any adult tissues tested (Fig. 5) with the exception of the ovary where precise SAS1B localization was observed only in oocytes of developing follicles (Fig. 5Q) and in rare oocytes identified suspended within the uterine lumen (black arrow in Fig. 5P). No immunoreactivity was observed in testis or epididymis. Similarly, the negative control PIM sera showed no immunoreactivity to any cell type in any of the tissue sections in the panel including oocytes within the ovary and uterus (data not shown). A commercially available pro-peptide human ASTL polyclonal antibody immunostained the oocytes (Fig. 5S) in the ovary in a pattern that was consistent with the other two antibodies used in this study. These identical findings using three different immunoreagents each generated to different SAS1B immunogens support an ovary-restricted and oocyte-specific pattern of SAS1B protein expression. Regardless of the Order Within Eutheria, SAS1B was Expressed Among Adult Ovarian Tissue Only in Ooplasm and Initiated Translation in Bilaminar Follicles Adult ovaries from seven eutherian species in four orders were included in this survey to investigate by IHC the temporal expression of SAS1B during folliculogenesis. The results are organized systematically under Order sub-headings. Using the GPgIM antibody generated to recombinant murine SAS1B, immunoreactivity was localized in ovarian sections (Fig. 6) from rat (Fig. 6B) and hamster (Fig. 6D) only within oocytes (black arrowheads for oocytes in secondary and later stages, red arrowheads for oocytes in transition follicles) beginning with oocytes residing within secondary follicles (Fig. 6B2) and continuing through all preantral (Fig. 6D2), early antral (Fig. 6B3) and Graafian follicle stages (Fig. 6D3). Oocytes within primordial and primary follicles did not express SAS1B protein (white arrowheads). No immunoreactivity to any cell type was seen in ovarian sections from either species using the GPgPIM (Fig. 6A1–3, C1–3). Figure 6Open in figure viewerPowerPoint SAS1B localization in Rodentia ovaries. IHC using GPgIM sera (generated to recombinant mouse SAS1B) on rat (B1–3) and hamster (D1–3) ovaries revealed strong brown immunoreactivity for SAS1B only in oocytes beginning with secondary follicles (B2) in both species and persisting in preantral (D2), early antral (B3) and Graafian follicles (D3) as seen in black arrowheads. None of oocytes in primordial and primary follicles showed any staining (white arrowheads). GPgPIM sera did not immunostain any cell type in any sections of rat (panel A1–A3) or hamster ovaries (panel C1–C3) including primordial, preantral and antral follicular stages. Magnification: ×200. SAS1B demonstrated oocyte specific and exquisite stage-specific localization, first appearing during folliculogenesis in oocytes that reside within follicles that had begun to form a secondary layer of granulosa cells and thus had begun the transition from primary into secondary follicles. This precise appearance of SAS1B translation in oocytes of secondary follicles was observed in both carnivore species studied and is well illustrated in the cat ovary in Figure 7B2 and B3 and in the dog ovary in Figure 7D1–3 using the GPgIM antibody. A classical primary-secondary transitional follicle with the oocyte surrounded by partial uni-laminar and partial bilaminar granulosa cell layers is illustrated in Fig. 7D1, demonstrating the initial appearance of SAS1B in this distinct morphological type of follicle. As a normal morphological feature, dog and cat ovaries are recognized to possess biovular follicles containing two oocytes surrounded by a single granulosa cell mass (gray arrowheads), a condition that, in the cat, may comprise 21% of follicles per ovary (Kennedy, 1924; Dederer, 1934). The absence of SAS1B staining in the biovular primary dog follicle in Figure 7D1, as well as the absence of SAS1B staining in oocytes of primordial/primary dog follicles (D1) was noted, with similar absences of SAS1B staining also noted at comparable early follicular stages in cat ovaries (Fig. 7B1). Figure 7Open in figure viewerPowerPoint SAS1B localization in Carnivora ovaries. Cat (A,B) and dog (C,D) ovaries were stained by IHC using the GPgIM (B,D) and GPgPIM (A,C) sera. Strong SAS1B immunoreactivity was noted only in oocytes (B2–B3: cats, D1–D3: dogs) including secondary (D2,D3) and antral follicles (B2,B3). Immunoreactivity was not observed in oocytes contained within primordial as well as primary follicles. A feature noted in all species examined and particularly well illustrated in Figure D1 was SAS1B staining in follicles that had initiated formation of a second layer of granulosa cells, the so called primary-secondary transitional follicle. In the primary to secondary transition follicle (D1) regions with a unilaminar layer of granulosa cells and regions with incomplete bilaminar morphology are denoted. SAS1B translation was first noted in oocytes of follicles at this primary-secondary transition stage of folliculogenesis (oocyte delineated by red arrowhead) and continued to be expressed in oocytes of secondary and tertiary follicles (oocytes denoted by black arrowheads, as in D2 and D3). Note biovular primary and primordial follicles are unstained (gray arrowheads, A1,C1). The PIM sera did not show any immunostaining of any cell types (panel A1–A3; C1–C3). Magnification: ×200 except for panel B3, ×400. In pig (Fig. 8A,B) and sheep (Fig. 8C,D) ovarian sections stained with GPgIM and PIM sera, SAS1B immunostaining was revealed only in oocytes contained in secondary and tertiary follicles (black arrowheads) with GPgIM sera. None of the oocytes in primordial and primary follicles reacted to the IM sera in either species (white arrowheads). PIM sera did not stain any cell types in either species. The sheep oocyte in Figure 8D2 was located within a Graafian follicle with a large antral cavity surrounded by a cumulus oophorus-oocyte complex and compact corona radiata. SAS1B protein localized in the ooplasm and was particularly concentrated in the oocyte cortex, a finding in concert with SAS1B\'s described role in mouse fertilization as an oolemmal receptor in ovulated oocytes (Sachdev et al., 2012). Figure 8Open in figure viewerPowerPoint SAS1B localization in Artiodactyla ovaries. Pig (A2, B2) and sheep (C2,D2) ovaries stained with the GPgIM sera, showed immunoreactivity in oocytes within secondary and tertiary antral follicles (black arrowheads) in both species. While immunoreactivity was not seen in oocytes contained within primordial or primary follicles (white arrowheads). The GPgPIM sera did not show immunostaining to any cell type in any pig (panel A1, B1) or sheep (panel C1, D1) ovarian sections. Magnifications: ×400 except for panels B1 and B2, ×200. Paired consecutive ovarian sections (Fig. 9) from the crab-eating macaque, Macaca fascicularis, were immunostained for SAS1B protein using Rb2IM (Fig. 9B) and PIM (Fig. 9A) sera. No appreciable immunostaining was seen on primate ovarian sections with the GPgIM antibody (which was made to recombinant mouse SAS1B) and thus the Rb2IM antibody (which was made to recombinant human SAS1B) was used. Distinct immunoreactivity was noted only in growing oocytes within secondary, preantral, early antral and Graafian follicles (Fig. 9, black arrowheads). The first definitive appearance of SAS1B occurred in nonhuman primate oocytes undergoing the primary- to-secondary follicle transition (red arrowhead in Fig. 9B3), although the intensity of the immunostain was not as vivid in these follicles as observed in oocytes within later follicular stages (compare Fig. 9B3 and Fig. 9B4). Oocytes residing within primordial and primary follicles did not immunoreact (white arrowheads); indicating the absence of SAS1B in the ovarian reserve and early growing oocytes while the PIM sera did not stain any cell type at any stage of macaque folliculogenesis. Figure 9Open in figure viewerPowerPoint SAS1B localization in nonhuman primate ovaries. Rb2PIM (A) sera revealed no SAS1B immunoreactivity to any cell type in ovarian sections spanning all stages of macaque folliculogenesis (A1–A6). In comparable sections to series A, strong SAS1B immunostaining was seen with the Rb2IM sera (B2–B6) only in oocytes. Immunoreactivity was not seen in oocytes within primordial (B1) or primary (B1) follicles (white arrowheads). The red arrowhead in B3 demarks staining in the ooplasm within a follicle at the primary-to-secondary transition, where the persistent unilaminar granulosa cell layer and incomplete bilaminar layers are noted. SAS1B persisted in macaque oocytes through preantral and secondary follicle stages (black arrowheads). Magnification: ×400. In the Rat Model, SAS1B Appeared First at Day 3 of Neonatal Life in Growing Oocytes That had Initiated a Bilaminar Layer of Granulosa Cells and Then Persisted Throughout Reproductive Life SAS1B was localized in rat ovaries (Fig. 10I) harvested during the following life stages: neonatal (days 0, 2, 3, 7), prepubertal (day 14), pubertal (day 28), adult (day 56), and retired breeders (180 days) by IHC using the Rb2IM antibodies (Fig. 10[I]A–H). The PIM control immunoreagent did not stain any cell type at any stage (small inset in respective aged panels). Absent in day 0 (Fig. 10[I]A) and day 2 (Fig. 10[I]B) ovaries, SAS1B protein was first detected as weak but definitive staining in day 3 neonatal oocytes within transitioning primary-secondary follicles (Fig. 10[I]C). The compact tunica albuginea of the day 3 ovarian cortex was densely populated with primordial follicles, which were SAS1B negative (Fig. 10[I]C). Distinctly larger (∼28 μm) than those within primordial follicles, the recruited clade of growing oocytes that resided within primary follicles was similarly SAS1B negative (Fig. 10[I]A–C). The intensity of SAS1B staining in neonatal ovaries increased in larger oocytes in preantral, antral and Graafian follicles. This stage specific pattern persisted in prepubertal, pubertal and adult ovaries (Fig. 10[I]E–H). SAS1B was restricted to growing oocytes that had reached the initiation of the bilaminar stage and beyond irrespective of the stage of reproductive life. In the ovaries of retired breeders (∼180 days) where fewer oocytes and follicles were observed, the pattern of SAS1B restriction to growing oocytes in secondary and subsequent follicular stages persisted. Figure 10Open in figure viewerPowerPoint (I). IHC localization of SAS1B protein in rat ovaries at neonatal, pubertal, adult and retired breeder stages. Panels A–H were stained with Rb2IM sera (Smaller inset images within each panel are aged matched sections that indicate no immunoreactivity seen with Rb2PIM sera). Panels A–D represent ovaries from neonatal female rats (ages 0, 2, 3, 7 days respectively); panel E, represents ovaries of prepubertal pups (age 14 days); panel F represents ovaries from pubertal animals (age 28 days), panel G represents adult rat ovary (age 56 days) and panel H represents retired breeder (age 180 days). SAS1B expression is seen only with IM sera. SAS1B was first expressed in ooplasm of oocytes in transition follicles beginning in day 3 ovary (as indicated by red arrowheads). This pattern of SAS1B staining persists throughout the life of the animal and irrespective of the age of the rodent, SAS1B was seen only in cytoplasm of oocytes in secondary follicles. No SAS1B was detected in oocytes housed primordial and primary follicles. Magnifications for panels were X100 except panels A6, B6 – A7, B7 were X200 and A8, B8 at X40. Rats were super-ovulated for oocyte retrieval and live oocytes were stained with the GPgIM or PIM antibody followed by a Cy3-labeled secondary antibody (Fig. 10[II]). SAS1B staining was most intense on one hemisphere of the oolemma in most oocytes. The ovulated MII oocyte in this figure reveals punctate SAS1B surface localization concentrated in a cap shaped domain corresponding to the microvillar region of the oolemma (red immunostain) which is known to lie antipodal to the eccentric MII arrested nucleus (blue stain) as seen in Fig. 10[II]A1 using the IM sera. No staining was observed with PIM sera as shown in Fig. 10[II]A2. SAS1B staining was observed on the membrane in macaque germinal vesicle (GV) oocytes and was more uniformly distributed throughout the egg surface (Fig. 10[II]B1 for IM and Fig. 10[II]B2 for PIM). Immunogold Labeling Shows SAS1B Concentrated in a Population of Cortical Granules Beneath the Oolemma The intracellular localization of SAS1B was studied in ultrathin sections of ovulated mouse cumulus oocyte complexes (COC) using immunoelectron microscopy (Fig. 12, inset of toluidine blue stain oocyte with extruded polar body marked by arrow in Fig. 12C). 6nM gold particles were concentrated on electron dense cortical granules (CG) of the MII oocytes using the Rb2IM sera (Fig. 12B,D). Control serial sections incubated with Rb2PIM sera showed an occasional gold particle localized in extra-cortical granule areas (Fig. 12A,C). Thus, SAS1B appeared to be most abundant in a sub-population of the most electron-dense cortical granules. Figure 11Open in figure viewerPowerPoint (II). SAS1B localization on rat and macaque oocytes. In rat MII oocytes, recovered after superovulation and live stained for SAS1B using GPgIM sera, SAS1B concentrated in a dome shaped domain on the surface of the rat oolemma (red stain in A1). The MII arrested oocyte nuclei (stained blue for DAPI in panels A1) were positioned eccentrically within the ooplasm antipodal to the SAS1B positive domain, indicating SAS1B staining was concentrated in the microvillar region. No SAS1B staining of oocytes was seen with GPgPIM sera as noted in panel A2. Macaque GV oocytes, fixed and then stained with RbIM sera showed a nonpolarized, uniform distribution on the oolemma (Panels B1). There was no staining with the RbPIM (panel B2). SAS1B (Astacin-like/ASTL/ovastacin), is a zinc metalloprotease which belongs to the M12A family (astacin family) of matrix metalloproteinases [EC 3.4.24.21] which contain a canonical HEXXHXXGXXH zinc metalloproteinase motif shared with the matrixins, adamalysins/reprolysins, and the serralysins (Stocker et al., 1995), protease families which with the astacins form a group which has been called the metzincin clan (MB) (Bode et al., 1993). Astacin was originally purified from the digestive tract of the freshwater crayfish, Astacus astacus and described as a Mr 11,000 endopeptidase (Pfleiderer et al., 1967). Subsequently the enzyme was shown to be zinc dependent (Stocker et al., 1988) and the designation astacin was introduced following the recommendation of the NC-IUBMB (Stocker et al., 1991; Dumermuth et al., 1991). Astacin-like digestive enzymes have been described in several species including the crab (Klimova et al., 1990). Astacin has become the prototype for a growing family of extracellular peptidases called the M12A astacin family (Bond and Beynon, 1995), now recognized in a wide variety of organisms and including several subfamilies of closely related members, such as the bone morphogenetic protein/tolloid-like enzymes, the meprins, and the hatching proteases. These M12A family enzymes target a wide range of substrates and participate in multiple biological processes such as early embryo patterning and morphogenesis in Drosophila, cartilage and bone formation and remodeling in vertebrates, digestion of dietary proteins in crayfish, and extracellular coat degradation during the course of embryo hatching in arthropods, birds, amphibians, and fish. For example, bone morphogenetic protein (BMP-1; a.k.a. procollagen C-proteinase), is an enzyme critical for assembly of collagen fibers (Suzuki et al., 1996). The meprins (A and B) are the closest relatives of SAS1B in their structural chemistry and relative position in the M12A family of metalloproteases. They were originally purified from mouse kidneys (Beynon et al., 1981) and shown to form high molecular weight oligomers anchored to the epithelial cell surface (Wolz and Bond, 1995), with meprin dimers apparently formed by disulfide bridges (Norman et al., 2003). SAS1B has also been referred as ovastacin because of its predominant expression in ovarian tissues (Quesada et al., 2004). As the structure and function of the SAS1B molecule becomes better understood, particularly the membrane isoforms, shared properties with other astacin family members may become evident. The present study demonstrates that SAS1B is oocyte specific in its pattern of translation across four orders of Mammalia. Two different immunoreagents against recombinant mouse (GPgIM) and human SAS1B protein (Rb2IM) demonstrated similar immunohistochemical staining profiles of ovary restricted translation and oocyte-specific localization in eight eutherians underscoring the reproducibility of the observations. These observations were confirmed with a commercially available anti-peptide immunoreagent to the ASTL pro-peptide domain. In all ovaries studied, SAS1B showed a tight temporal and spatial onset of translation at the primary-secondary follicle transition with SAS1B translation persisting in growing and mature ovulated oocytes. This pattern of appearance of SAS1B at the primary-secondary follicle transition occurred in ovaries of neonatal, prepubertal, pubertal and adult animals and the pattern was not affected by the stage of the estrus cycle. Figure 13A summarizes the timing and staging of various known oocyte/germ cell proteins during folliculogenesis and places the SAS1B translation pattern in the context of a developmental window encompassing formation of primordial follicles through development of growing secondary follicles. The time-line references embryonic or post-natal days of mouse oogenesis. The temporal expression of previously characterized genes is illustrated based on protein or RNA analyses including transcription factors such as Figla (Millar et al., 1993; Soyal et al., 2000), Sohlh1 (Pangas, 2006), Nobox (Rajkovic et al., 2004), RNA binding proteins such as Dazla (Ruggiu et al., 1997), Ybx2 (Gu et al., 1998), ECAT1 (unpublished data), and MOEP19 (Herr et al., 2008); factors which function synergistically to maintain quiescence of primordial follicles such as PTEN (Reddy et al., 2008) and TSc/mTORC1 (Adhikari et al., 2010); the zona proteins ZP1, ZP2, ZP3 (Phillips and Shalgi, 1980); growth factors such as Gdf9 and BMP15 (McGrath et al., 1995, Dube et al., 1998); the maternal effect gene Mater (Tong et al., 2004); heat shock protein HSP90 (Vanmuylder et al., 2002; Pires and Khole, 2009); egg and embryo-abundant peptidylarginine deiminase-like protein ePAD (Wright et al., 2003) and the cortical granule protein ovoperoxidase, present in the growing follicles, that is activated upon fertilization (Gulyas and Schmell, 1980). Each of these proteins has been reported to have a precise timing for onset of expression. Notably, SAS1B is the first protein reported to initiate translation only in oocytes at the primary to secondary follicle transition. Based on our initial survey of several eutherian orders it may be inferred that SAS1B can serve as a biomarker for oocyte entry into the primary-secondary transition follicular stage in many species. Figure 12Open in figure viewerPowerPoint Ultrastructural localization of SAS1B proteins in a sub-set of electron dense granules in the cortex of mouse MII oocytes. Insert in panel (C) shows a toluidine blue stained MII oocyte from a gonadotrophin-stimulated superovulated mouse used for the sectioning and staining of ultrathin images. Note extruded polar body (arrow within the perivitelline space, PVS). Rare gold particles immunoreacted with cortical granules or other organelles using the PIM sera and secondary gold-conjugate (A & C). However, intense SAS1B immunostaining revealed by 6 nm gold particles concentrated on electron dense cortical granules (CG, black arrowheads) lying within the ooplasm close to the ruffled membrane of the oocyte using the RbIM sera (B & D). Figure 13Open in figure viewerPowerPoint A: Schematic diagram of SAS1B translation during oocyte growth and folliculogenesis compared with other oocyte/germ cell specific biomarkers. The time line refers to embryonic (E) or post natal (PD) days of mouse ovary development. Periods of expression of well-known proteins are illustrated based on protein or RNA analyses reported in the literature including Figla, Sohlh1, Nobox, Dazla, Ybx2, ECAT1, MOEP19, PTEN, TSc/mTORC1, zona proteins ZP1, ZP2, ZP3, Gdf9, BMP15, MATER, HSP90, peptidylarginine deiminase-like protein ePAD, and ovoperoxidase. A unique feature of SAS1B is its appearance in growing oocytes at the primary-to secondary follicle transition. B: Model of SAS1B expression stages and implications for biomarker development and as a target for a contraceptive ovastatic/ovalysin. SAS1B is expressed only in oocytes (brown color) in follicles that have initiated formation of bilaminar granulosa cells and in subsequent stages. This metalloprotease may therefore serve as a target for an ovalysin that selectively acts on growing oocytes while sparing the pool of reserve primary oocytes within primordial and primary follicles. SAS1B is an oocyte-specific protein and its membrane accessibility may allow delivery of targeted drugs that deliver ovastatic/ovalytic payloads. To date, six reports have characterized various aspects of SAS1B/ovastacin biology and presented evidence for physiological functions that include roles in several steps of fertilization and early development. The first report by Quesada et al. (2004) suggested that ovastacin in mammals could function in blastocyst hatching similar to known hatching proteases in evolutionary distant species including arthropods and fish (Geier and Zwilling, 1998). On the other hand, Sachdev et al. (2012) defined a role for SAS1B as a oolemmal binding partner for the sperm intra-acrosomal ligand SLLP1 during sperm binding and fusion suggesting that the name SAS1B, sperm acrosomal SLLP1 binding, best conveys these biological interactions at the time of fertilization. The absence of SAS1B messages and proteins in blastocysts led Sachdev et al. (2012) to conclude also that the proposed role for SAS1B in hatching from the zona pellucida in mammals was unlikely. In birds, a study by Acloque et al. (2012) demonstrated that astl is dynamically expressed in embryonic stem cells and embryonic epithelium during morphogenesis suggesting an important function for the control of epithelial cell behavior during early chick embryo development. Burkart et al. (2012) localized the SAS1B/ovastacin protein in mouse cortical granules by immunofluorescence microscopy, presented biochemical evidence that this metalloprotease cleaves the zona pellucida [ZP] protein-ZP2, and proposed a role for SAS1B in zona hardening leading to the block to polyspermy that ensures monospermic fertilization and successful development (Burkart et al., 2012). A study by Peng et al. (2012) identified ASTL by LC-MS/MS in the media from ZP intact MII mouse oocytes that had been activated by 10 mM SrCl2 to induce exocytosis, further confirming that a population of SAS1B is secreted from granules located at the oocyte cortex. Strontium is a parthenogenetic agent for mouse oocytes that induces repetitive intracellular calcium releases in a manner similar to those following normal fertilization by spermatozoa. A recent study by Dietzel et al. (2013) demonstrated that fetuin-B inhibits ovastacin and sustains fertility, leading to a model in which plasma fetuin-B levels are predicted to restrain SAS1B protease activity and thereby maintain ZP permeability until after gamete fusion. Given that three temporally distinct events in the fertilization cascade and in early development and hatching have been proposed as functions for SAS1B, it is important to consider the possibility that the SAS1B protein is multifunctional and/or that different protein isoforms encoded by the astl gene are engaged in different functions. The astl gene in mouse ovary undergoes alternative splicing involving at least six distinct mRNAs, resulting in SAS1B protein isoforms ranging from 31 to 62 kDa (Sachdev et al., 2012). It is possible that some of these isoforms are membrane bound while others are secreted and that selected SAS1B isoforms engage in the different biological functions such as sperm-oolemmal interactions and fertilization as well as in ZP2 proteolysis and the block to polyspermy. At present, the assignment of different functions to specific SAS1B splice variants is entirely unknown and an area of active research interest. SAS1B may ultimately be categorized as a \"moonlighting protein” where multiple functions of ASTL splicing add a striking dimension to protein microheterogeneity and cellular complexity to benefit cells with distinct but synergistic functional isoforms (Jeffery, 1999). The demonstration of SAS1B in this study in several Mammalian orders and its tight temporal regulation during oogenesis in each of these species suggests the molecule likely plays an important role in fertilization across Mammalia. With respect to SAS1B\'s proposed function at the time of sperm-oocyte binding during fertilization, SAS1B was the first molecule reported to be an oolemmal binding partner for a defined sperm ligand, thus revealing a sperm-oocyte binding partner pair (Sachdev et al., 2012). Several other candidates have been proposed to be binding partners during fertilization, such as oocyte proteins that are members of the tetraspanin family (CD9, CD81), GPI-anchored oocyte proteins, PIG-A, and sperm epididymal protein DE (CRISP1); however, these proteins have been validated as candidate partners only for the sperm-oocyte fusion process (Coonrod et al., 1999; Miyado et al., 2000; Cohen et al., 2000; Alfieri et al., 2003). Proteins in the multi-membered ADAM family such as fertilin α (ADAM1), fertilin β (ADAM2), and cyritestin (ADAM3) have not qualified to be binding partners but have other putative reproductive biology roles (Almeida et al., 1995; Evans et al., 1995, 1997; Yuan et al., 1997; Cho et al., 1998; Shamsadin et al., 1999; Nishimura et al., 2001). The targeted deletion of the sperm inner-acrosomal protein Izumo results in infertility due to a block in sperm internalization into the oocyte (Inoue et al., 2005); however a binding partner for Izumo on the egg membrane has not yet been identified. Thus, SAS1B holds considerable importance as a defined oolemmal binding partner for an intra-acrosomal sperm ligand during mammalian fertilization. The IHC results of the present study failed to detect SAS1B protein in any nonovarian tissue with the exception of the uterus, where only rare oocytes free in the uterine lumen were immunostained. This pattern of expression restricted to the ovary and oocyte confirms bioinformatics data from heat map analyses of expression of astl mRNAs in adult mouse tissues as well as mouse embryonic stages that may be obtained by interrogating the expressed sequence tag datasets [ESTs] in GenBank [http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.31313]. The results of the present study are in accord with those of Quesada et al. (2004), who showed oocyte restricted concentration of astl messages by in situ hybridization in mice, and Sachdev et al. (2012), who demonstrated ovary specificity at the transcript level in mice by Northern blot and oocyte restriction at the protein level on mouse ovary sections by IHC. The observed expression patterns of SAS1B lead to the conclusion that ASTL is under tight temporal and spatial gene control. Not only was SAS1B expressed solely in oocytes and only at a precise stage of oogenesis in all the species tested but this translation pattern occurred in follicles that had begun formation of bilaminar granulosa cell layers in neonatal, prepubertal, pubertal, adult and retired breeder rats. SAS1B expression in rats began at day 3 of life in oocytes that had initiated bilaminar follicle formation and this expression continued in a similar manner irrespective of the age of the animal (Fig. 10[I]C–H). In addition, when ovaries were harvested at various stages of the estrus cycle in mice; the expression of SAS1B remained un-changed in any of the estrus stages (Fig. 4) suggesting that ASTL is not under the control of known steroid hormones or other reproductive hormones regulating estrus. The correlation of SAS1B translation to formation of the bilaminar granulosa cell layer opens exciting avenues for the future studies of gene regulation during folliculogenesis. Western blot analysis of ovarian extracts from different mammals showed immunoreactive bands of 44–46 kDa to be the dominant SAS1B isoforms (with additional immunoreactive bands seen in some species). The data in Figure 3 indicate that SAS1B protein in mouse shows ∼45 and 52 kDa forms (Fig. 3A1); rat ∼45 and 55 kDa forms (Fig. 3B1); cat ∼44 kDa form (Fig. 3C1); dog ∼37, 46, 55, 75 kDa forms (Fig. 3D1); pig ∼46 kDa form (Fig. 3E1); sheep ∼44 kDa form (Fig. 3F1); and macaque ovarian extracts show a ∼46 kDa form (Fig. 3G1). None of the preimmune sera showed any immunoreactivity to any protein/s in any of the respective ovarian extracts (Fig. 3A2–G2). The variation in forms and sizes could be due to splice variants/isoforms of the protein and/or various posttranslational modifications of the processed protein. Higher immunoreactive bands could be dimers or trimers of the protein. Studies of the protein isoforms derived from individual splice variants are areas for future exploration. Comparison of SAS1B protein sequences in several of the test species confirms the proteinase domain is the most conserved region. This domain houses the enzyme\'s active catalytic site and the putative transmembrane domain. The catalytic region is 100% conserved whereas there is ∼86% conservation in the transmembrane domain. Notably, the c-terminus seems to be species specific with the least conserved amino acid residues. It is this region in which species specific interaction domains may reside. The present study highlights the prominence of SAS1B across all the eutherians tested (Figs. 6-9) strongly suggesting that, as in the mouse, SAS1B could play a vital role in the sperm-oocyte binding process and block to polyspermy in other species. Phylogenetic trees based upon multiple sequence alignments of proteins from many species are commonly used to determine the evolutionary relationships between homologous sequences, which can give insights into the evolution of a protein family and the functional specificity of the members of the family (Zuckerkandl and Pauling, 1965). Bioinformatic analysis of SAS1B conservation indicates that there is 70–96% identity of amino acids in the protein sequence in the Eutherians tested; the macaque being the closest and identical to the human protein sequence (Fig. 2). In all the species tested, it was observed that the protein appears in a precise stage in oogenesis and folliculogenesis. Because the pattern of expression is consistent it may be predicted that the expression pattern noted in macaque ovaries is similar to that of human ovary as well as other mammals with SAS1B translation appearing in bilaminar follicles and continuing until the oocytes are ovulated. Live staining of oocytes collected after a superovulation protocol in mice (Sachdev et al., 2012) and in rats in the current study, revealed staining of SAS1B at the cell surface predominantly localized to the well-developed microvillar domain on the egg plasma membrane. In contrast, in species without a well-developed microvillar domain, such as primates (Santella et al., 1992), SAS1B showed a uniform distribution on the oolemma in macaque GV oocytes. The findings from our results on IIF indicate that the macaque GV oocyte does not have a distinct microvillar domain where microvilli are concentrated, but rather microvilli in the macaque GV oocyte are more sparsely and uniformly distributed on the oolemma. These findings lead to the prediction that irrespective of species, a population of SAS1B is accessible on the surface of the oocyte, a conclusion that is in concert with both its proposed roles as an oolemmal receptor for the sperm (Sachdev et al., 2012) and as a ZP2 cleaving enzyme involved in the block to polyspermy (Burkart et al., 2012). Ultrastructural Localization of SAS1B in Cortical Granules of Mouse MII-arrested Oocytes Electron microscopic immunolocalization of SAS1B in ovulated oocytes (Fig. 12A) showed high concentrations of antibody associated with a population of electron-dense cortical granules in the peripheral ooplasm. Cortical granules become competent to undergo exocytosis before ovulation. Fertilization triggers cortical granule migration to the plasma membrane, where they fuse and exocytose their contents (Wessel et al., 2001; Ducibella et al., 1988, 2002). Burkart et al. (2012) took advantage of the unique C-terminal extension of ovastacin (SAS1B) and generated a peptide-specific rabbit antibody which was used to stain SAS1B in ovulated oocytes by fluorescence microscopy. The lectin LCA-FITC (Lens culinaris agglutinin conjugated to FITC), which binds α-linked mannose residues, served as a biomarker of cortical granules. SAS1B signals colocalized with LCA-positive granules by immunofluorescence microscopy at the periphery of ovulated oocytes indicating the presence of ovastacin within cortical granules. Although the study demonstrated LCA-positive granules, ovastacin was not localized ultrastructurally. When sperm contact the egg membrane at the stage of sperm binding, events involving oocyte surface SAS1B and sperm SLLP1 molecules and other un-identified binding partners initiate membrane fusion and the phagocytic processes that lead to sperm internalization and eventual syngamy. Sperm fusion triggers the cortical granule reaction involving calcium dependent and SNARE protein-mediated exocytosis, one result of which is the block to polyspermy (Liu, 2011). The total number of cortical granule proteins is not definitively known. However, Liu (2011) estimated 4–14 distinct proteins are contained in these granules in picogram quantities per oocyte. Previous studies using polysaccharide-binding dyes and reporters revealed cortical granules to be rich in carbohydrate moieties (Selman and Anderson, 1975), proteinases (Hoodbhoy and Talbot, 1994), ovoperoxidase (Gulyas and Schmell, 1980), calreticulin (Munoz-Gotera et al., 2001), N-acetylglucosaminidase (Miller et al., 1993), p32 (Gross et al., 2000), and peptidylarginine deiminase (Pierce et al., 1990). Liu (2011) described a small population of granules that are exocytosed before sperm penetration, while several granule components are also reported to function following fertilization in regulating embryonic cleavage and preimplantation development. Thus cortical granules may be a heterogeneous group of organelles with differing content and the identification of SAS1B in the population of electron dense cortical granules may help to distinguish this cohort. Timely release of these granules is essential for a successful fertilization to occur, especially for individuals who are reproductively challenged and require in vitro fertilization. Previous studies together with the present data (Fig. 12) lead to the conclusion that a population of cytoplasmic SAS1B molecules is packaged into vesicles that form electron-dense cortical granules. The finding of SAS1B both within cortical granules and on the oolemma at the time of fertilization, when the microvillar domain functions in sperm binding, are consistent with a model in which cortical granules undergo exocytosis fusing with the oolemma to integrate a population of SAS1B into the oolemma and also release granule contents into the perivitelline space. Manipulation of fertility, by means of novel drugs or immunocontraception, has long been the subject of intense research. Immunocontraception today encompasses contraceptive vaccines, which may interfere with gamete production, fertilization, or embryogenesis by stimulating an immune reaction to key reproductive molecules (Nation et al., 2008), and passive immunization, usually in the form of antibodies or antibody-drug conjugates to reproductive targets. Before development of a new contraceptive drug or immunocontraceptive key questions need to be addressed regarding target validation, particularly the specificity of expression of the target in reproductive organs (Aitken et al., 2008). For small molecule drug approaches, the target should be druggable: for example ion channels, enzymes, and receptors contain defined domains and biologically active clefts that may interact with small molecule inhibitors. As the immunohistochemical data in this study demonstrate, one diagnostic application for the SAS1B biomarker lies in the follicular staging of growing oocytes. Future clinical chemistry approaches with immunometric assays might also determine if SAS1B concentrations can be measured in serum, saliva or blood, and if SAS1B concentrations might predict the presence of a pool of growing oocytes. The SAS1B protein might thus offer an improved means to verify that a population of growing oocytes is present in an animal. Such a diagnostic assay will likely need to have high sensitivity but, if achieved, might predict menarche before it occurs, detect oocyte growth following chemotherapy (and thus obviate the need to maintain oocyte cryopreservation), verify oocyte growth in cases of infertility (to better diagnose the etiology of infertility), or predict candidates suitable for ovarian stimulation and IVF. Contraceptive targets in the ovarian follicle associated with the oocyte, somatic cells or both have been considered. For example, Anti-Mullerian hormone (AMH) plays important roles in both primordial follicle activation and antral follicle selection. Recombinant AMH and/or agonists have been proposed as contraceptive strategies to block recruitment of primordial follicle (Grootegoed and Themmen, 2001). Advancements in recombinant technology have allowed the production of bio-activated AMH (Weenen et al., 2004). However, the EST database reveals that AMH is not unique to the ovary but is also present in several other tissues such as Sertoli cells of the testes, brain, embryonic tissues, eyes, lungs, mammary glands, prostate, stomach, and uterus (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.112432). Growth differentiation factor 9 (GDF9) holds promise as candidate contraceptive target due to its essential requirement for oocyte maturation. Ovaries from GDF-9-deficient female mice demonstrate that primordial and primary unilaminar follicles can be formed, but there is a block in follicular development beyond the primary one-layer follicle stage which leads to complete infertility (Dong et al., 1996). GDF9 is expressed in oocytes and granulosa cells of primary follicles (Aaltonen et al., 1999). Immunocontraceptive studies in sheep have found that immune responses generated against a peptide vaccine corresponding to 1–15 amino acid residues on the N-terminus of GDF9 cause anovulation in ewes following primary and single booster vaccinations (McNatty et al., 2007). Finally, Bone Morphogenic Protein 15 (BMP15) is a growth factor that remains constant in expression throughout folliculogenesis, being detected initially in oocytes and granulosa cells in the primordial follicle stage in the human ovary (Aaltonen et al., 1999; Teixeira Filho et al., 2002; Margulis et al., 2008), in primary follicle stage oocytes in the mouse (Dube et al., 1998), and in primordial follicle oocytes in the sheep (McNatty et al., 2001) suggesting a species specific role for BMP15. Notably, EST databases indicate that BMP15 is not only expressed in ovary but also in tissues such as skin, joints, and lung, indicating it may not offer a reproductive tissue-restricted contraceptive target. The SAS1B protein fulfills several key requirements of a contraceptive target for a small molecule inhibitor, a targeted immunocontraceptive, or a contraceptive vaccinogen. (1) SAS1B is tissue specific: SAS1B transcripts and protein are only observed in the ovary and are absent in other adult tissues (Fig. 5). (2) SAS1B is involved in key events at the time of fertilization. (3) SAS1B is an oolemmal target: (Fig. 10[II], and Sachdev et al., 2012) and is prominent on the microvillar domain where fertilization occurs in species with this feature. (4) SAS1B contains one or more \"drugable” domains: SAS1B is an active metalloprotease and has an active site which can be drugged or targeted by small molecule/inhibitors. (5) In adult ovaries, SAS1B is absent in the ovarian reserve of quiescent oocytes: thus drugs and antibodies targeted to SAS1B are predicted to affect only growing oocytes and to preserve the ovarian reserve, affording a reversible mechanism of contraceptive drug action. Together these biological characteristics of SAS1B suggest this molecule may be a suitable target for novel nonsteroidal contraceptive approaches. A model depicting the mechanism of action of a SAS1B contraceptive drug/inhibitor/vaccine is diagramed in Figure 13B. Because the expression of SAS1B is precise viz., it first appears only in growing oocytes of follicles undergoing the primary-secondary transition the action of a contraceptive targeted against SAS1B might be directed only to growing oocytes in developed follicles. Because SAS1B is not expressed in the ovarian germ cell reserve residing in primordial follicles, contraceptive reversibility and the restoration of fertility would be predicted to occur through the pool of resting oocytes replenishing the wave of folliculogenesis. Targeted deletions of SAS1B in mice by two groups (Sachdev et al., 2012; Burkart et al., 2012) showed reduced fertility, although not complete infertility. These results suggest that targeting SAS1B with a small molecule antagonist that acts to inhibit SAS1B enzymatic function may not be a strategy to achieve high contraceptive efficacy in primates. However, the oocyte specific expression and cell surface localization of SAS1B indicate this molecule deserves consideration for reversible nonhormonal contraceptive strategies particularly immunological approaches. As a contraceptive vaccinogen, SAS1B may be capable of evoking complement fixing antibodies which may initiate complement mediated cytoxicity reactions or induce cytotoxic T cell mediated oophoritis, mechanisms that may arrest or lyse the oocyte or prevent progression of oocyte maturation or ovulation. Vaccination with the oocyte proteins that comprise the zona pellucida have been shown to lower fertility rates in a wide range of animals but their use in primates has been hampered by a broad effect on folliculogenesis and concerns about maintaining menstrual cyclicity (Grootenhuis et al., 1996). Thus an effective contraceptive strategy in primates may be imagined whereby oocyte specific effects are induced, possibly blocking oocyte maturation or oocyte fertilizability while follicular maturation and corpora luteal formation remain unimpaired. Crucial to determining the feasibility of such a strategy using SAS1B will be an understanding of exactly when during folliculogenesis SAS1B becomes accessible at the cell surface and which SAS1B epitopes are exposed. Passive immunization with an anti-SAS1B antibody-drug conjugate (ADC) also may be envisioned as a novel contraceptive approach. The liquor folliculi is a transudate of the serum and contains IgG and IgA immunoglobulin concentrations at least 50–60% that of serum but only 10% of serum IgM (El-Roeiy et al., 1987; Mantzavinos et al., 1993, Hammadeh et al., 2002). Thus passively administered antibodies or antibody-drug conjugates, perhaps an intramuscular depot of IgGs, are predicted to be able to reach the lumen of antral follicles and bind to the oolemma by diffusion through the cumulus mass. Other nonimmunological approaches may also be conceived. If a substrate can be identified that is selective for the SAS1B enzymatic pocket, a peptide-drug conjugate might deliver a cytostatic or cytotoxic payload selectively to the growing oocyte by means of SAS1B\'s cell surface accessibility. Thus, the findings of this study envision a class of contraceptives that target the oocyte or early embryo to selectively arrest oocyte and early embryo development. Based on SAS1B\'s biological features in several mammalian orders a new type of contraceptive is envisioned termed an \"oostatin” representing a reimagined goal of contraceptive research. Human SAS1B (hSAS1B) gene-specific primers were designed to create an NdeI site at the 5′ end and a HindIII site at the 3′ end of PCR amplicons (NCBI accession no. NM_001002036). Primers bearing restriction sites were obtained from GIBCO BRL (Life Technologies, Carlsbad, CA) (forward primer: 5′-CAT ATG TAA ATG ATT CCT GCA ATT AAC CAA GGG-3′, reverse primer: 5′-AAG CTT GGA AGC TAG GGT CTG A-3′). PCR was performed with 0.2 ng of human ovarian cDNA (BD Biosciences-Clontech, San Jose, CA) in a 25 μl assay system in a MJ Research (Waltham, MA) thermal cycler using a program of one 1 min cycle at 95°C followed by 34 cycles of denaturation, annealing and elongation at 94°C for 45 sec, 58°C for 45 sec and 72°C for 90 sec, respectively, and a final elongation step at 72°C for 5 min. The hSAS1B amplicons were separated on a 1% NuSieve (FMC Bio-Products, Rockland, ME) agarose gel, subcloned into the PCR 2.1-TOPO vector (Invitrogen, San Diego, CA), and sequenced in both directions to confirm authenticity, using vector-derived and insert-specific primers. The primers amplified region 163 bp to 1,104 bp (942 bp) of the 1,296 bp mRNA corresponding to amino acids 55–368, yielding a truncated version of the protein with a mass of 314 amino acids; ∼35 kDa. This protein contained domains conserved among species including the catalytic domain. Expression and Purification of Human SAS1B/ Recombinant Protein and Antibody Production hSAS1B was cloned into the pET28b+ expression vector and constructs were transformed into the E. coli expression host, BL21(DE3) (Invitrogen), containing a chromosomal copy of the T7 RNA polymerase gene under the control of IPTG-inducible lacUV5 promoter. Bacterial production and immobilized metal affinity chromatography (IMAC) purification of recombinant protein containing a C-terminus His-tag was performed as previously described (Westbrook et al., 2000). The IMAC-purified rec hSAS1B was concentrated using a Centricon10 micro concentrator (Amicon, Beverly, MA) and then purified using the prep cell system (Model 491, Bio-Rad). Protein fractions corresponding to sharp elution peaks were tested by Western blotting with anti-His-tag antibody (SIGMA, St. Louis, MO) and fractions positive with the antibody were pooled, lyophilized, and reconstituted in a minimum volume of PBS. Protein concentrations were determined using the BCA method according to the manufacturer\'s specifications (Pierce, Rockford, IL). Purified recombinant SAS1B protein was analyzed by SDS-PAGE followed by silver staining and homogeneity of the protein was confirmed by mass spectrometry where data was acquired on an LTQ-XL instrument using LC coupled with MS/MS. Male rabbits were immunized by means of sub-cutaneous injections at multiple sites with purified human SAS1B protein. The immunization protocol comprised an initial dose of 100 μg in 500 μl of PBS emulsified with 500 μl of Freund\'s Complete Adjuvant (Sigma-Aldrich, St. Louis, MO). Two booster vaccinations followed at 15-day intervals, each containing 100 μg of the immunogen in 500 μl of PBS in the presence of 500 μl of Freund\'s Incomplete Adjuvant (Sigma-Aldrich). Blood samples were collected from the medial auricular artery, before the initial injection (preimmune control), 7–10 days after booster injections, and at 3 monthly intervals throughout the remaining collection period. A final blood sample was collected by means of cardiac puncture as rabbits were anesthetized with ketamine/xylazine 50/5 mg per kg body weight by intramuscular injections followed by isoflurane by mask. Blood samples were allowed to clot and then centrifuged at 4,000 rpm for 10 min. Clear sera were separated, aliquoted, labeled, and stored at −80°C for subsequent analysis. Inbred Belgium white adult rabbits, Swiss mice, hamsters, and Holtzman rats were housed in a temperature-controlled room with a 12-hr light cycle. These studies were conducted under protocol 2545 approved by the Institutional Animal Care and Use Committee at the University of Virginia in accord with the humane use of animals in research. Cat, dog tissues were obtained from animals undergoing surgical oophorectomy at the Charlottesville-Albemarle County Society for the Prevention of Cruelty to Animals. Pig tissues were obtained from animals undergoing surgical oophorectomy at the University of Virginia. A sheep ovary was obtained from a local farmer in Charlottesville at the time of slaughter. Macaque tissues (Macaca fascic) were obtained from the National Primate Research Center at the University of Washington in Seattle as a part of a collaborative study. Rabbit polyclonal antibody (Rb2IM) to human SAS1B protein (as described above), a previously produced guinea pig polyclonal antibody (GPgIM) to full length mouse SAS1B (Sachdev et al., 2012), and a commercially available rabbit polyclonal antibody to the pro-peptide domain of human ASTL (Abcam, Cambridge, MA) were used in this study. Fab\'-specific horse-radish peroxidase labeled (HRP) secondary antibodies (anti-rabbit, anti-guinea pig; Jackson Immunoresearch) were used for immunohistochemistry, ELISA, and Western blotting. For indirect immunofluorescence anti-rabbit Alexafluor conjugates (Molecular Probes) and anti-guinea pig Cy3 conjugates (Jackson Immunoresearch) were used. For ultrastructural immunolocalization, 6-nm gold particles tagged with goat anti-rabbit secondary antibody (Electron Microscopy Sciences, Hatfields, PA) were used. Titers of polyclonal antibodies (against recombinant human SAS1B) in the sera of human SAS1B-immunized rabbits were determined by ELISA, with PIM samples serving as negative controls for each rabbit. Assays were performed in 96-well ELISA plates (Nunc, Denmark) using 100 ng of recombinant SAS1B as the target antigen in 100 μl of bicarbonate-carbonate buffer pH 9.6. Nonspecific binding was blocked with 5% nonfat dry milk in PBS (NFDM-PBS). Serial dilutions of the primary antibodies starting at 1:500 were used. Antibodies were incubated for 1 hr at 37°C and unbound antibodies were removed by washing wells 5 times with PBS containing 0.05% (v/v) Tween-20 (Sigma). This was followed by detection with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody diluted 1:5,000 in NFDM-PBS, with incubation at 37°C for 1 hr. The substrate tetramethyl benzidine (TMB/ H2O2; Sigma-Aldrich), which reacts with HRP, produced a blue reaction product. The enzyme reaction was stopped by adding 1 N HCl and optical density was measured at 490 nm on a Titerteck Multiscan plate reader (Biotek, USA). Endpoint antibody titers were expressed as the highest dilution at which the absorbance of immune sera remained greater than the absorbance of preimmunization sera. Comparisons of the ASTL genomic and SAS1B protein sequences from the available deposits for Pan troglodytes, Macaca mulatta, Bos Taurus, Canis familiaris, Mus musculus, Rattus norvegicus, Drosophila melanogaster were generated from the NCBI database on HomoloGene. The available CDS sequences for the above species were submitted on EMBL-EBI ClustalW2- Phylogeny for generation of a phylogenetic tree (http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/). Adult rats were injected intraperitoneally with 10 IU pregnant mare serum gonadotropin (PMSG) (Sigma) to stimulate follicular development and 48 hr later with 10 IU of human chorionic gonadotropin (hCG) (Sigma) to induce ovulation. Sixteen to 18 hr after hCG injection, rats were exsanguinated after cervical dislocation and oocytes were harvested from the oviducts in fresh M16 medium (Invitrogen) within 5 min of dislocation directly into media under oil. Cumulus cells were separated from cumulus-oocyte complexes (COC) using 0.05% hyaluronidase in M16 and oocytes were allowed to recover for 2 hr at 37°C. Guinea pig PIM and IM sera were diluted in M16 at a 1:200 dilution and incubated with oocytes for 2–3 hr at 37°C. Oocytes were washed 5 times with medium and then fixed in 4% (w/v) para-formaldehyde for 15 min. Following three washes in PBS for 5 min each, oocytes were incubated with a 1:500 dilution of goat anti guinea pig Cye3 conjugate to which was added 0.1% (w/v) DAPI (Roche, USA) to counterstain nuclei and incubated for 1 hr at room temperature. Excess antibody and dye were removed by washing thrice with PBS for 5 min each. Oocytes were mounted on well-slides in slowfade (Molecular Probes) and immunostaining was observed at 200–400× magnifications by phase and fluorescent microscopy (Axio Vert 200, Carl Zeiss, Germany). Macaque ovaries were obtained from the Washington National Primate Research Center tissue distribution program and transported to the laboratory at 37°C in mHTF+BSA within 1 hr of collection. Cumulus-oocyte complexes were retrieved by ovarian dissection and puncture of 3 to 6 mm follicles into mHTF+BSA at 37°C. Immature COC were identified with a visible germinal vesicle (GV) under a dissecting microscope and treated with hyaluronidase (40 IU/ml) to assist with removal of cumulus cells by gentle pipetting. Cumulus free GV oocytes were prepared for immunostaining as described above but using Rb2IM and PIM at a 1:50 dilution and goat anti- rabbit Alexa 568 secondary antibody at a dilution of 1:500 and DAPI at 5 μg/ml concentration followed by imaging on fluorescent microscopy. Tissues were harvested and lysed in CELIS buffer with protease inhibitor cocktail (Celis et al., 1992). Proteins were transferred from unstained gels to nitrocellulose membranes using a Bio-Rad Trans Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer\'s instructions. Membranes were blocked with NFDM-PBS for 1 hr at room temperature and incubated overnight at 4°C with a 1:1,000 dilution of guinea pig or rabbit PIM and IM sera. After washing thrice with PBS-Tween (0.05% Tween-20 in PBS), the membranes were incubated with HRP-conjugated goat anti-rabbit/guinea pig immunoglobulin (Jackson Immunoresearch labs, USA) at 1:5,000 dilution, washed × 3, and immunoreactive bands were detected by ECL (GE healthcare Life Sciences, Buckinghamshire, UK) or were visualized with the colorimetric reagent 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (KPL, Gaithersburg, MD). Specimens were fixed by immersion in Bouin\'s fixative for 24–36 hr including mouse tissues (brain, pancreas, thymus, thyroid, skeletal muscle, heart, kidney, liver, lung, spleen, intestine, testes, epididymis, ovary, oviduct, and uterus), and ovaries of rat, hamster, cat, dog, pig, sheep, and macaque. Ovaries of female neonatal rat pups (aged day 0, 3, 7) were fixed for 12 hr, and day 14, 28, 56, and 180 day ovaries were fixed for 24–36 hr. Tissues were dehydrated in a graded series of ethanol, embedded in paraffin, and 4–5 μM sections collected on glass slides. The specimens were melted at 56°C, deparaffinized in xylene, quenched for endogenous peroxidase activity in methanol-hydrogen peroxide and rehydrated. Sections were blocked with NFDM containing 5% normal goat serum, for 2 hr at room temperature. The slides were then incubated overnight at 4°C with a 1:100 dilution of guinea pig polyclonal antibody to SAS1B (Sachdev et al., 2012) / rabbit PIM or IM serum (1:100) as indicated in the figure legends. The rabbit polyclonal to human SAS1B was used to stain macaque sections, mouse multiple tissue IHC and rat ovaries in the ontogeny IHC study; all other specimens were immunoreacted with the guinea pig antibody to mouse SAS1B. After washing thrice, the slides were be incubated with 1:500 dilution of HRP-conjugated goat anti-guinea pig/ anti-rabbit immunoglobulin. After 5 washes, the immunoperoxidase products were developed using 3,3′-diaminobenzidine (DAB) chromogen solution (SIGMA), then counterstained for 2–3 min using hematoxylin, dehydrated through series of graded alcohols, cleared in xylene, and mounted in DPX mounting medium. Vaginal swabs were collected using a cotton tipped swab wetted with ambient temperature physiological saline and inserted into the vagina of the restrained mouse. The swab was gently turned, rolled against the vaginal wall, and then removed. Cells were transferred to a dry glass slide and air dried, overlaid with a coverslip and viewed immediately at 200× magnification under bright field illumination. The stage of the estrous cycle was determined based on the presence or absence of leukocytes, cornified epithelial, and nucleated epithelial cells (Felicio et al., 1984). Mice were studied for three estrus cycles before collection of ovarian tissue for IHC using GPgIM and PIM antibodies as described above. Mouse cumulus-oocyte-complexes (collected as described in the gamete collection section) were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde with 3% sucrose in 0.1 M sodium phosphate, pH 7.4. After washing with 0.1 M phosphate they were centrifuged into 10 μl of 3% gelatin in 0.1 M phosphate that had been melted and then cooled to room temperature. The gelatin was hardened at 4°C and the remainder of the dehydration and Lowicryl K4M (Electron Microscopy Sciences, Hatfields, PA 19440) embedding steps were performed following the manufacturer\'s protocol at 4°C. Ultrathin sections were cut with a Reichert-Jung Ultracut E microtome, collected on 300 mesh nickel grids and then air dried overnight at room temperature. Nonspecific binding was blocked with an incubation buffer (IB) solution of 5% normal goat serum and 5% BSA in PBS, phosphate buffered saline, pH 7.4, for 1 hr at room temperature. Grids were then incubated with the Rb2IM or PIM antibodies diluted 1:60, respectively, in IB with 0.2% Aurion BSA-c in PBS (IB) (Electron Microscopy Sciences, Hatfield, PA 19440). Incubation in primary antibodies was overnight in a humidified chamber at 4°C. Grids were then washed 5 times for 5 min each with IB followed by 2 hr incubation at room temperature with the Aurion goat anti-rabbit IgG secondary antibodies coupled to 6 nM gold particles (Electron Microscopy Sciences, Hatfields, PA 19440). After washing with IB and distilled water, grids were stained with 5% uranyl acetate in 50% ethanol, carbon coated, and examined with a JEOL 1230 electron microscope equipped with a digital camera. The authors thank Ms. Sheri Vanhoose and Ms. Virginia Rubianes in the UVA Histology Core; and Ms. Gina Wimer from the Department of Comparative Medicine for assistance with histology and animal work, respectively. Dr. Ken Victor and Mr. Charles Lyons from Pathology assisted with mass spectrometry protein microsequencing. Thanks to the Charlottesville-Albemarle County Society for the Prevention of Cruelty to Animals for providing us un-used tissues from their neutering program in support of research towards nonsurgical pet population control. Thanks to Mr. Hlavin for providing us with sheep ovarian tissue for this study. This project received funding from NIH Fogarty International Center, NIH ARRA supplement from the Contraceptive Development Branch, a Grand Challenges Exploration Grant from the Bill & Melinda Gates Foundation, a grant from the Wallace H Coulter Translational Research Partnership Endowment, and a grant from the Paul Mellon Urologic Oncology Institute at the University of Virginia Cancer Center. This research was also supported in part by NIH and ITHS to the National Primate Research Center at the University of Washington. 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