A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), can directly associate with gametes or with the early embryo in the oviduct. Although the glycoprotein is widely distributed among mammalian species and there is indirect evidence concerning the involvement of the molecule in the fertilization process, its physiological functions are far from completely understood. To understand the fundamental mechanisms that direct gene expression as well as to know the physiological significance of OGP, we have isolated and characterized a mouse OGP gene (mogp-1). The gene was found to span 13.4 kilobases (kb) including 11 exons and 10 introns. The genomic organization of mogp-1 is well conserved compared to the other members of the chitinase family. Two transcription initiation sites were found at positions 18 and 14 upstream from the first ATG codon. Fluorescence in situ hybridization analysis demonstrated that the mogp-1 was located on the R-positive F3 band of mouse chromosome 3. Although the putative promoter region of mogp-1 lacked typical TATA, CAAT, or GC box sequences, the region contained several motif sequences of transcription factor binding sites including 10 half-palindromic estrogen responsive elements (ERE) and an imperfect ERE. Transient transfection experiments demonstrated that promoter activity could be modulated by various sequences within the 2.2 kb of the 5′-flanking region, and that the mogp-1 promoter was transactivated in an estrogen receptor-positive cell line, MCF-7, by the addition of estradiol-17β (E₂). In addition, relevant promoter activity for E₂ responsiveness resides within the first 270 base pairs upstream of the mogp-1. These findings should facilitate our understanding of the regulation of OGP gene expression, and they may be helpful for designing experiments to unravel the role of OGP in the process of mammalian fertilization.

We evaluated five practical diets in which 0%, 25%, 50%, 75%, and 100% (dietary treatments 1–5) of fish meal protein was replaced by solvent-extracted cottonseed meal protein. Adult rainbow trout (initial average weight 247 ± 8 g) were fed the diets over a period of 131 days during which a general 2-fold body weight increase occurred. The total diet gossypol concentration (free and protein-bound) showed a gradual increase with increased cottonseed meal substitution. Blood samples were collected on Days 0, 64, 112, and 131 for hematological and steroid hormone determination in plasma of males and females. Hemoglobin content was significantly reduced in fish from treatment 5 (7.9 ± 0.3 g/dl) in comparison to treatments 1–3 (10.3–10.9 g/dl). After 112 and 131 days of feeding, testis weights, concentrations of testosterone, and 11-ketotestosterone were elevated in fish from dietary treatments 2 and 3 in comparison to control and diets 4 and 5. On Day 71, sperm were collected from 6 fish per dietary treatment to assess sperm quality. No significant differences in sperm concentrations (7.2–9.8 × 10⁹/ml), motility (78–89%), and standardized (300 × 10⁵ sperm/egg) fertilizing ability (18.9–22.6% hatched embryos) were found. Total gossypol concentrations in blood plasma differed significantly among treatments, and the levels were among the highest ever recorded in animals fed cottonseed-supplemented diets (2.9 ± 0.2, 11.7 ± 4.1, 21.7 ± 1.4, and 29.9 ± 3.9 μg/ml, for treatments 2–5, respectively). The major portion of gossypol in blood plasma was protein-bound (81–93%). This was in contrast to minute amounts of gossypol present in seminal plasma, mostly in free form (0.02–0.18 μg/ml), which indicates the presence of a barrier between general circulation and the testis with respect to gossypol distribution in lower vertebrates. Thus, the reproductive parameters of male rainbow trout examined in this study were not significantly affected by feeding cottonseed meal for 131 days.

Continuous cultures of bovine trophectoderm (CT-1 and CT-5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro.

Detrimental effects of oxygen-derived free radicals on embryos during culture have been demonstrated in several species. Vitamin E occurs naturally in cell membranes and protects cells from oxidative stress. Under some conditions, vitamin C acts synergistically to enhance the antioxidant effects of vitamin E, a benefit that may be further enhanced by EDTA. The present experiments concerned culture of bovine embryos derived from in vitro-matured, fertilized oocytes with vitamin E, vitamin C, and EDTA in a chemically defined culture medium 0.2% BSA at 5% O₂, 5% CO₂, and 90% N₂. In the first experiment, more zygotes developed to expanded blastocysts (17%, n = 224, P < 0.05) when culture medium contained 100 μM vitamin E than in control medium (11%, n = 234). Development to early, expanded, and hatched blastocysts was lower with vitamins E and C combined than with vitamin E alone (15%, 9%, and 2% vs. 24%, 17%, and 5%, respectively; P < 0.05), as was the mean number of cells per blastocyst (56 vs. 84, P < 0.05). Addition of EDTA (3 μM) failed to improve development over that in culture with vitamin E vitamin C. In experiment 2, in vitro-produced embryos cultured 5.5 days in medium with or without 100 μM vitamin E were transferred nonsurgically to recipient cows and heifers and then collected nonsurgically 7 days later. Embryos cultured with vitamin E (n = 37) were approximately 63% larger in surface area than controls (1.16 mm² vs. 0.71 mm² surface area; n = 27, P < 0.04).

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20–30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.

While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of N^ω-nitro-l-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with γ-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.

We tested the hypothesis that progesterone (P₄) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) ± P₄ (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P₄ levels by RIA. Aminoglutethimide inhibited P₄ synthesis by > 95% and increased the level of apoptosis as evidenced by ³²P-labeled oligonucleosomal DNA fragmentation (> 40%). P₄ supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P₄ has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P₄ was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in ³²P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P ≥ 0.05) in P₄ levels after treatment with PR antagonists. These observations support the concept that P₄ represses the onset of apoptosis in the CL by a PR-dependent mechanism.

Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P₄) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P₄ on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.

The objective of this study was to examine longitudinal changes in serum leptin concentrations during development and to correlate those changes with sexual development in male rhesus monkeys housed under natural environmental conditions. Blood samples were drawn from 8 control animals approximately every other month from 10 to 30 mo of age and thereafter monthly through 80 mo of age. Leptin levels declined through the juvenile period until the onset of puberty and were negatively correlated with body weight. Seven of the eight animals became sexually mature during the breeding season of their fourth year of life. Puberty was delayed in the other animal until the subsequent breeding season. There were no significant fluctuations in leptin levels prior to or in association with the pubertal rise in LH and testosterone (T) secretion. During the peripubertal period, levels of leptin varied between 2 and 3 ng/ml. The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period. With the achievement of sexual maturity, leptin levels varied seasonally, with peak levels in the late winter (Jan–Mar) and a nadir in the late summer (Aug–Sept). A late winter rise in leptin was also evident in most of the animals during Years 2 and 3, but not during Year 4. In the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3–4 mo behind the seasonal increase in LH and T. In the fall of Year 5, but not thereafter, leptin levels were positively related to percent body fat and negatively correlated with lean body mass. The data do not support the hypothesis that increasing leptin concentrations trigger the onset of puberty in the male rhesus monkey. During the juvenile period and after sexual maturation, but not during the peripubertal period, leptin secretion varied with season in the animals; but the environmental factors that cue or drive this rhythm remain to be determined.

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24–48 h of culture when 100 μM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34^cdc2 kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 μM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34^cdc2 kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34^cdc2 kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34^cdc2 and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).

Previous research demonstrated that sperm mobility, i.e., the net movement of a sperm population, is a quantitative trait of the domestic fowl. However, the cellular basis for this trait was unknown. In the present work, individual motile sperm were evaluated with a Hobson SpermTracker in order to identify one or more properties of motile sperm that could account for variation in sperm mobility observed among males. A method was validated for assessing sperm motion over an erythrocyte monolayer at body temperature. A small-scale experiment with roosters from the tails and center of a normal distribution of sperm mobility phenotypes (n = 33 roosters) demonstrated that straight line velocity (VSL) and motile concentration were critical to expression of phenotype. The importance of these variables was confirmed with a large-scale experiment using a representative subpopulation (n = 100 roosters). VSL of individual sperm at 41°C ranged between 5 and 100 μm/sec. VSL averaged 32, 39, and 40 μm/sec for low, average, and high sperm mobility phenotypes. Sperm were diluted to 1.2 × 10⁶/ml for motion analysis. Mean motile concentrations were 0.52, 0.84, and 0.95 × 10⁶/ml for low, average, and high sperm mobility phenotypes. Motile concentration was correlated with sperm mobility (r = 0.71). VSL appeared to have an additive effect as it was correlated with straightness of sperm cell trajectory (r = 0.79).

By the use of ribonuclease protection assay (RPA) combined with immunohistochemical techniques, the expression of estrogen receptor (ER) α and ERβ was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ERα mRNA was detected at all ages examined; ERβ mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERβ in differentiating granulosa cells of the ovary, whereas ERα was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ERα mRNA at all ages examined with little or no significant levels of ERβ, except on PND 1 when a low level of message appeared. In males, ERα and ERβ mRNA were detected in the fetal testis; however, ERβ gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ERα in the interstitial compartment of the testis, whereas ERβ was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP₁₂, was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab′ antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP₁₂ peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP₁₂ Fab′ antibodies recognized a protein band of ∼72 ± 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP₁₂ that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.

The present study was undertaken to identify the mechanisms underlying the effect of insulin-like growth factor I (IGF-I) on FSH receptor (FSHR) in rat granulosa cells. Treatment with FSH produced a substantial increase in FSHR mRNA level, as was expected, while concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced FSHR mRNA, with a maximal response 2.8-fold greater than that induced by FSH alone. IGF-I, either alone or in combination with FSH, did not affect intracellular cAMP levels, whereas it enhanced the effect of 8-bromo (Br)-cAMP on FSHR mRNA production. Taken together, these findings suggest that the ability of IGF-I to enhance FSH action concerning the induction of FSHR is exerted at sites distal to cAMP generation. We then investigated whether the effect of IGF-I and FSH on FSHR mRNA levels was the result of increased transcription and/or altered mRNA stability. The rates of FSHR mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-I. On the other hand, the decay curves for the 2.4-kilobase (kb) FSHR mRNA transcript in primary granulosa cells significantly altered the slope of the FSHR mRNA decay curve in the presence of IGF-I and increased the half-life of the FSHR mRNA transcript. These data suggest a possible role for changes in FSHR mRNA stability in the IGF-I-induced regulation of FSHR in rat granulosa cells. Treatment with activin produced a substantial increase in FSHR mRNA level, as was expected, and concurrent treatment with IGF-I did not affect activin-induced FSHR mRNA. Our data suggest that the IGF-I effect on FSHR expression is related to cAMP production induced by FSH and may maintain FSHR mRNA level because of prolonged FSHR mRNA stability.

Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca² -regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1–5 mm) and medium-sized (5–8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells.

Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-β1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-β1 dose-response studies revealed an ED₅₀ of 0.24–0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion.

Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca² -sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 μM) stimulated intracellular free calcium ion concentrations ([Ca² ]_i) in single porcine theca cells. The [Ca² ]_i elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca² ]_i response.

Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-β and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.

Telomere DNA at the physical termini of chromosomes forms a single-stranded 3′ overhang. In lower eukaryotes, e.g., ciliated protozoa, this DNA extension is capped by specific proteins that have been structurally and functionally characterized. Much less is known about single-stranded telomere DNA-binding proteins in vertebrates. Here we describe a new protein from bovine sperm designated bsSSTBP that specifically interacts with single-stranded (TTAGGG)_N DNA. The bsSSTBP was extracted from nuclei by 0.6 M KCl. The native size of this protein, estimated by gel filtration, was 20–40 kDa. SDS-PAGE of the UV cross-linked complex between bsSSTBP and telomere DNA indicated that several polypeptides are involved in complex formation. Bovine sSSTB had high specificity toward nucleotide sequence, since single nucleotide substitutions in the (TTAGGG)₄ substrate suppressed binding. The minimal number of (TTAGGG) repeats required for binding of bsSSTBP was 3, and the protein recognized linear but not folded DNA structures. We propose that the bsSSTBP participates in telomere-telomere interactions and the telomere membrane localization observed in mature sperm. In mammals, somatic telomere-binding proteins are apparently substituted by sperm-specific ones that may lead to a structural reorganization of telomere domains to fulfill functions important during meiosis and fertilization.

Short day lengths or reduced food availability are salient cues for small mammals that breed seasonally. Photoperiod-mediated gonadal regression in white-footed mice (Peromyscus leucopus) is a slow, orderly process that involves testicular apoptosis. Testicular regression in response to restricted caloric intake is relatively rapid, and it is generally reversed quickly by ad libitum (ad lib) feeding. To determine the contribution of apoptotic cell death during food restriction, and to examine possible interactions with photoperiod, mice housed in long (16L:8D) or short (8L:16D) photoperiods were fed either ad lib or 70% of their average ad lib intake. Testes were removed at 2, 4, 6, or 8 wk of experimental treatment. Apoptotic activity was determined by in situ TUNEL labeling and assessment of DNA laddering. Significant (P < 0.05) gonadal regression in response to short days was first detected at 8 weeks in mice fed ad lib. Food-restricted, long-day mice also showed significant testicular regression at 8 wk. Combined exposure to short day lengths and food restriction resulted in significant testicular regression at 6 wk (P < 0.05). TUNEL labeling was slightly, though significantly, elevated in germ cells at 2 and 4 wk in long-day food-restricted mice (P < 0.05). TUNEL labeling was also elevated in short-day food-restricted males at these early times but then increased nearly 5-fold at 6 and 8 wk in these mice (P < 0.001). DNA laddering confirmed elevated apoptosis. Overall apoptotic activity negatively correlated with paired testis mass, plasma testosterone, and spermatogenic index measurements in both ad lib and food-restricted males. Few histological markers of necrotic cell death were observed in any group. Taken together, these results suggest that testicular regression in response to limited caloric intake or short days is mediated by apoptosis.

The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO₂, 7% O₂, 88% N₂ culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Thirty ovariectomized sows were used in an experiment designed to determine whether the ability of the porcine uterus to release prostaglandin (PG) F_2α in response to oxytocin is regulated by progesterone (P₄) and estradiol (E₂). Sows were assigned to one of four treatment groups: 1) no steroids (ovariectomized controls; n = 8), 2) E₂ (n = 8), 3) P₄ (n = 7), or 4) E₂ P₄ (n = 7). P₄ and E₂ were administered so as to mimic the normal temporal changes that occur in these hormones during the estrous cycle. A group of intact sows (n = 9) was included for comparison. All sows received an injection of oxytocin (30 IU, i.v.) on Days 12, 15, and 18 postestrus. Jugular venous blood samples were collected from 60 min before through 120 min after injection of oxytocin for quantification of 13,14-dihydro-15-keto-PGF_2α (PGFM). Preinjection baseline concentrations of PGFM, the magnitude of the PGFM response above baseline, and area under the PGFM response curve (AUC) were calculated for each sow on each day and compared among treatment groups by ANOVA. Among the ovariectomized sows receiving steroid replacement, baseline concentrations of PGFM were low on Day 12 postestrus in all four groups. On Days 15 and 18, baseline concentrations remained low in the two groups that did not receive P₄ but increased in those that did. Both the magnitude of the response to oxytocin and AUC were small on Day 12 postestrus in all 4 groups. By Day 15, the magnitude of the response and AUC increased in the group that received both P₄ and E₂ but remained low in the other three groups. By Day 18, responses to oxytocin were greater in both groups that received P₄ than in those that did not. Baseline concentrations were similar in intact sows and in those that received both P₄ and E₂ on all three days examined. The magnitude of the response and the AUC were greater in the ovariectomized sows receiving P₄ and E₂ replacement than in the intact control sows on Days 15 and 18 postestrus. From these results, we conclude that P₄ and E₂ interact to control the time when the uterus begins to secrete PGF_2α in response to oxytocin and the amount of PGF_2α secreted.

In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.

Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of β-cyclodextrin (βCD) as cholesterol acceptor. After incubation with 8 mM βCD, 50–60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.

The objective of this study was to determine the effect of embryo production systems on the expression of insulin-like growth factor (IGF)-II mRNA in fetal bovine tissues at Day 70 of gestation (63 days after transfer). Oocytes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. Zygotes were cultured in either tissue culture medium (TCM)-199 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or TCM-199 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 h postinsemination, IVPSR embryos were transferred into fresh TCM-199 10% ECS whereas IVPS embryos had fresh medium replaced. All embryos were cultured for an additional 96 h. In vivo-produced embryos were harvested from superovulated Holstein cows (multiple ovulations [MO]). Grade 1 blastocysts from all groups were transferred singly into Angus heifers. At Day 70 of gestation, fetuses (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were collected; liver and skeletal muscle samples were snap frozen, and whole-cell RNA (wcRNA) was extracted. Levels of IGF-II mRNA were determined by RNase protection assay and quantified relative to 18S rRNA (mean arbitrary units ± SEM). WcRNA from adult and Day 90 fetal bovine liver were used as controls. Adult liver contained 9-fold less IGF-II mRNA than liver from Day 90 fetuses (P < 0.05). Fetal livers of males originating from IVPS and IVPSR groups possessed approximately 2-fold greater levels of mRNA for IGF-II than those from MO males (0.25 ± 0.07, 0.33 ± 0.04, and 0.14 ± 0.03, respectively; P < 0.05). Levels of mRNA for IGF-II tended to be lower (P = 0.07) in skeletal muscle of fetuses originating from the IVPSR group (0.043 ± 0.005) compared to MO controls (0.070 ± 0.008). In conclusion, at Day 70 of gestation, fetuses originating from in vitro production systems possessed altered levels of IGF-II mRNA in both liver and skeletal muscle.

In this differential-display polymerase chain reaction-based study, four different primer sets generated cDNA fragments of ovarian carbonyl reductase genes that were uniquely expressed during the ovulatory process in eCG-primed immature rats. The temporal pattern of expression of this aldo-keto reductase gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation via injection of the primed animals with hCG. The results showed that at least four homologous forms of this gene were transcribed during ovulation. Northern blot analyses indicated a 14-fold increase in ovarian mRNA for carbonyl reductase, with expression reaching a peak at 8 h after hCG treatment and then declining to negligible levels during the next 16 h. In situ hybridization revealed that most of the transcription was in the thecal connective tissue of the ovary and was absent from the granulosa layer of ovarian follicles. Treatment of the animals with ovulation-blocking doses of epostane (an inhibitor of progesterone synthesis) or indomethacin (an inhibitor of prostanoid synthesis) did not reduce the expression of ovarian carbonyl reductase. Nevertheless, the temporal pattern of expression of carbonyl reductase after the induction of ovulation suggests that this enzyme activity is at least indirectly associated with the ovulatory process.

The superoxide radical and its scavenger, superoxide dismutase (SOD), play important roles in the regulation of corpus luteum function. The present study was undertaken to investigate whether SOD is related to pregnancy-induced maintenance of corpus luteum function. Placentae obtained from rats on Day 12 of pregnancy were incubated for 24 h, and the supernatant was used as placental luteotropins. Pseudopregnant rats were given the placental incubation medium from Day 9 to Day 12 of pseudopregnancy. The treatment significantly increased serum progesterone concentrations on Day 12 of pseudopregnancy. Both activities and mRNA levels of copper-zinc SOD (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in the corpus luteum were also increased on Day 12 of pseudopregnancy. Treating the placental incubation medium with charcoal significantly eliminated the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD expression, but not on Cu,Zn-SOD expression. The inhibitory effect of the charcoal treatment on luteal Mn-SOD expression was reversed by supplementation with testosterone or dihydrotestosterone (DHT), but serum progesterone concentrations were recovered only by DHT. Testosterone or DHT alone had no effect on serum progesterone concentrations and luteal SOD expression. In conclusion, placental luteotropins increased SOD expression in the corpus luteum and stimulated progesterone production, suggesting that SOD is involved in the maintenance of the corpus luteum function by placental luteotropins. In addition, androgen, with other placental luteotropins, acted to stimulate progesterone production and Mn-SOD expression in pseudopregnant rats.

Eosinophils are present in human endometrium only immediately before and during menstruation, suggesting a role in that process. The expression of the eosinophil chemoattractant, eotaxin, and its receptor, CCR3, within the human endometrium were investigated by immunohistochemical analysis of tissue sections spanning the entire menstrual cycle. Eotaxin was localized to perivascular cells in the late secretory phase, and it was also identified in eosinophils. However, the highest levels of this chemokine were present in both luminal and glandular epithelial cells during the proliferative and secretory phases of the cycle. Treatment of endometrial tissue with monensin, which blocks protein secretion, increased epithelial immunoreactive eotaxin, substantiating synthesis in these cells. Although the CCR3 receptor was expressed by eosinophils, it was also strongly expressed by endometrial epithelial cells. The CCR3 receptor on purified, cultured endometrial epithelial cells was functional, as assessed by a transient Ca² flux in response to eotaxin. These analyses demonstrate that eotaxin is expressed by endometrial cells and may therefore be involved in the recruitment of eosinophils into this tissue premenstrually. However, the observation that this chemokine and the CCR3 molecule are strongly expressed by epithelial cells throughout the cycle suggests that these proteins may have additional important functions within the endometrium.

The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6–73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24–72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9–80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 ± 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 ± 5.8% of the cells at the end of G1 stage and 37.0 ± 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.

An up-regulated cDNA fragment was obtained from differential-display polymerase chain reaction of brook trout ovarian tissue stimulated by phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187. Using this cDNA as a probe, a full-length cDNA of 2267 base pairs was obtained by screening a library of PMA/A23187-stimulated ovarian cDNA. The mRNA obtained presumably encodes for a 302-amino acid protein showing similarities with several members of the tumor necrosis factor (TNF) receptor superfamily. The protein contains several cysteine-rich domains characteristic of mammalian TNF receptor members and is most similar to human decoy receptor 3 and osteoprotegerin, two soluble decoy TNF receptors. Consequently, this TNF receptor homologue was tentatively named a trout decoy receptor (TDcR). On Northern blots of ovarian tissue, TDcR hybridized with a 2.2-kilobase transcript that was strongly up-regulated under phorbol ester stimulation. TDcR mRNA was localized in granulosa cells and was detected in the ovary during and after natural ovulation. Its expression was up-regulated at the end of ovulation and progressively down-regulated after 48 h postovulation. Among other trout tissues tested, the transcript was present only in the testis. To our knowledge this is the first description of a member of the TNF receptor family from a lower vertebrate and the first report of a decoy-like TNF receptor in the vertebrate ovary.

Oxytocin receptors in myometrium of women, rats, and rabbits rise markedly before the onset of labor, suggesting a role in the initiation of labor. In guinea pigs, a previous study reported no such rise by one-point determination of oxytocin binding. The purpose of this study was to use a more rigorous method to determine whether the binding characteristics of myometrial oxytocin receptors change in relation to labor in guinea pigs. Competitive binding studies were carried out in microsomes from inner and outer myometrium between 42 days of gestation and labor. Binding to analogs was also tested. Data were analyzed with affinity spectra and LIGAND. Oxytocin bound to one site with a dissociation constant of 6.3 ± 0.65 × 10⁻⁹ M. Binding capacity was 1.0 ± 0.1 × 10⁻¹² mol/mg protein. The Hill coefficient was near unity. No significant changes occurred with gestation or labor in dissociation constant, binding capacity, or Hill coefficient (all P ≥ 0.2, nested ANOVA). Binding capacity was higher in the outer than in the inner layer (1.2 ± 0.2 vs. 0.8 ± 0.1 × 10⁻¹² mol/mg protein, P = 0.02), but the dissociation constants were similar. Differences existed in the dissociation constants of the analogs tested. The main conclusion is that oxytocin receptors are unlikely to have a regulatory role in the initiation of labor in guinea pigs.

Hypothalamo-pituitary disconnected Soay rams were exposed to two photoperiodic treatments: 1) constant long days (16L:8D) for 48 wk after pretreatment under short days (LD group), and 2) constant short days (8L:16D) for 48 wk after pretreatment under long days (SD group). In the LD group, plasma prolactin (PRL) concentrations increased from 0 to 8 wk (maximum: 143.3 ± 8.4 μg/l; 8.8 ± 1.2 wk), decreased from 9 to 34 wk (minimum: 15.6 ± 1.6 μg/l; 34.5 ± 1.5 wk), and finally increased again under the constant conditions, with a similar cyclical pattern for all individuals. In the SD group, PRL concentrations showed an inverse pattern (minimum: 8.6 ± 2.6 μg/l; 17.1 ± 2.0 wk; maximum: 46.4 ± 5.5 μg/l; 30.2 ± 3.2 wk), with more variability. Plasma concentrations of FSH were basal in both groups. The duration of the daily nocturnal melatonin peak (measured at 10, 24, and 44 wk) remained close to 8 h under long days (high-fidelity melatonin signal) but decreased significantly (13.8 h to 9.3 h) under short days (low-fidelity melatonin signal). The results support the conclusion that the melatonin signal encoding photoperiod acts within the pituitary gland to induce both acute (inductive) and chronic (refractory) effects photoperiod on PRL secretion.

Angiogenesis is fundamental for human endometrial development and differentiation necessary for implantation. These vascular changes are thought to be mediated by the vascular endothelial growth factor (VEGF), whose specific receptors have not been examined in detail thus far. We conducted the present study to determine, by immunocytochemistry and computerized image analysis of the functionalis, the expression and modulation of the receptors Flk-1/KDR and Flt-1, which mediate VEGF effects on endothelial mitogenicity, chemotaxis, and capillary permeability. VEGF receptors are expressed mainly in endometrial endothelial cells, with variations of intensity and number of stained capillaries related to the phase of the cycle. The number of capillaries immunostained for Flk-1/KDR was maximal in the proliferative phase (ratio Flk-1/CD34: 1), twice as high as the number of Flt-1-expressing capillaries (ratio Flt-1/CD34: 0.47). The staining intensity for Flk-1 decreased during the late proliferative and early secretory phases, to increase again in the midsecretory period. The number of Flt-1-labeled capillaries was about 2-fold higher in the secretory than in the proliferative phase; however, the proportion of Flt-1-positive cells did not change, owing to the associated increase in vascular density that characterizes progression of the functionalis from the proliferative to the secretory stage. The staining intensity for Flt-1 was higher during the late proliferative and secretory phases (especially in the midsecretory phase) and the premenstrual period. In contrast, the proportion of capillaries expressing Flk-1/KDR decreased in the secretory phase (ratio Flk-1/Von Willebrand factor: 0.55). Enhanced expression of Flk-1/KDR, and of Flt-1, on narrow capillary strands at the beginning of and during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration following menstrual shedding. The high coexpression of Flk-1/KDR and Flt-1 observed on capillaries during the midsecretory period correlates with an increase of endometrial microvascular density and of permeability characteristic of this phase of the cycle, which is a prerequisite for implantation. Finally, strong expression of Flt-1, but not Flk-1/KDR, was observed on dilated capillaries during the premenstrual period and the late proliferative phase, suggesting preferential association of Flt-1 with nonproliferating capillaries at those times; activation of this receptor by VEGF could be involved in premenstrual vascular hyperpermeability, edema, and extravasation of leukocytes. In addition to the endothelial localization, we found that epithelial cells expressed Flt-1 and Flk-1/KDR. We conclude that Flt-1 and Flk-1/KDR in the functionalis are modulated in parallel or independently according to the phase of the cycle, and that these changes are responsible for VEGF actions on endometrial vascular growth and permeability. The molecular mechanisms concerning these regulations will require further investigation.

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F_2∝ (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.

The purpose of this study was to evaluate the impact of angiotensinogen gene (Agt) deficiency on reproductive fitness in a rodent model. Mice with 0 (Agt^−/−), 1 (Agt^−/ ), and 2 (Agt ^/ ) copies of Agt were bred according to the following schemes: 1) Agt^−/− × Agt^−/−, 2) Agt^−/ × Agt^−/ , 3) Agt ^/ × Agt ^/ , and 4) Agt ^/ ♀ × Agt^−/ ♂. There were 4 breeding pairs per scheme. Breedings were time mated. Mice and litters were weighed daily. Southern blotting was used for genotyping. We found that Agt^−/− breeding pairs had fewer litters (2 [range 1–2] vs. 4 [range 3–5]; P = 0.01), fewer pups per litter (4 [range 1–7] vs. 6 [range 1–10]; P = 0.006), and longer interpregnancy intervals (43 days [range 31–44] vs. 35.5 days [range 22–58]; P = 0.04) compared to wild-type controls. The ratio of postcoital plugs to subsequent litters was 4.0 and 1.2 for Agt^−/− and Agt ^/ breedings, respectively (P = 0.03). Median maternal weights during all trimesters of pregnancy were significantly lower for Agt-deficient mice compared to wild-type controls. Among Agt^−/ × Agt^−/ breedings, the proportions of Agt ^/ (n = 17), Agt^−/ (n = 38), and Agt^−/− (n = 4) offspring differed significantly from the expected 1:2:1 Mendelian inheritance pattern (P = 0.03). Neonatal survival among the offspring derived from the Agt^−/− × Agt^−/− breeding scheme was significantly reduced (P = 0.001). We conclude that Agt deficiency is associated with an in utero lethal effect, decreased fertility, and impaired neonatal survival.

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection.

In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 μm² of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 μm²) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively.

In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40–50 nm) and bovine herpesvirus-1 (BHV-1; 180–200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.

To date, cloned farm animals have been produced by nuclear transfer from embryonic, fetal, and adult cell types. However, mice completely derived from embryonic stem (ES) cells have been produced by aggregation with tetraploid embryos. The objective of the present study was to generate offspring completely derived from bovine ES-like cells. ES-like cells isolated from the inner cell mass of in vitro-produced embryos were aggregated with tetraploid bovine embryos generated by electrofusion at the 2-cell stage. A total of 77 embryo aggregates produced by coculture of two 8-cell-stage tetraploid embryos and a clump of ES-like cells were cultured in vitro. Twenty-eight of the aggregates developed to the blastocyst stage, and 12 of these were transferred to recipient cows. Six calves representing 2 singletons and 2 sets of twins were produced from the transfer of the chimeric embryos. Microsatellite analysis for the 6 calves demonstrated that one calf was chimeric in the hair roots and the another was chimeric in the liver. However, unfortunately, both of these calves died shortly after birth. Two of the placentae from the remaining pregnancies were also chimeric. These results indicate that the bovine ES-like cells used in these studies were able to contribute to development.