Search

The male reproductive system develops from a minimally functioning gonad and nonfunctioning accessory sex glands in the neonate; sex steroids, presumed to be primary influencers of these changes, have been characterized in multiple species. This study focused on the expression of the androgen receptor as the principal mediator of androgen-induced signaling; the 5α reductase enzyme that converts testosterone to the more active dihydrotestosterone; and colony stimulating factor 1, a mediator of macrophage influence on organ development in the pig. The time points chosen to evaluate normal developmental changes during the juvenile and prepubertal intervals included the inflection time points of 6.5 weeks of age at the nadir of circulating estradiol and testosterone concentrations in juveniles, and 11 weeks of age, when these concentrations begin to increase. The role of sex steroid signaling in the regulation of gene expression was evaluated by the blockade of androgen and estrogen receptors and reduction in endogenous estrogens. Expression of colony stimulating factor 1 in the testes gradually decreased during development; developmental profiles in the prostate and seminal vesicles were clearly different. Interference with sex steroid signaling had no effect on the expression of these three genes in testicular tissue and minimal and transient effects in prostate and seminal vesicles.

Intranasal oxytocin (IN OXT) administration has been proposed as a pharmacological treatment for a range of biomedical conditions including neurodevelopmental disorders. However, studies evaluating the potential long-lasting effects of chronic IN OXT during development are still scarce. Here we conducted a follow-up study of a cohort of adult titi monkeys that received intranasal oxytocin 0.8IU/kg (n=15) or saline (n=14) daily for six months during their juvenile period (12 to 18months of age), with the goal of evaluating the potential long-lasting behavioral and neural effects one year post-treatment. Subjects were paired with an opposite-sex mate at 30months of age (one year post-treatment). We examined pair affiliative behavior in the home cage during the first four months and tested for behavioral components of pair bonding at one week and four months post-pairing. We assessed long-term changes in brain glucose uptake using 18FDG positron emission tomography (PET) scans. Our results showed that OXT-treated animals were more affiliative across a number of measures, including tail twining, compared to SAL treated subjects (tail twining is considered the "highest" type of affiliation in titi monkeys). Neuroimaging showed no treatment differences in glucose uptake between SAL and OXT-treated animals; however, females showed higher glucose uptake in whole brain at 23months, and in both the whole brain and the social salience network at 33months of age compared to males. Our results suggest that chronic IN OXT administration during development can have long-term effects on adult social behavior.

The number of Sertoli cells has a major effect on adult testis size and sperm production capacity. Mechanisms that regulate the number of Sertoli cells in livestock are at best nebulously understood; however, with lesser testicular estrogen production, proliferation of Sertoli cells is prolonged compared with vehicle-treated littermates. Decreased WISP2 gene expression in testes as a result of less endogenous estrogen is similar to altered WISP2 gene expression following corticosteroid treatment of some cultured cells. Taken together, these findings indicate decreased testicular cortisol might be in the signaling pathway between reduced endogenous estrogens and the prolonged interval of Sertoli cell proliferation. Hence, in these studies, potential actions of testicular corticosteroid on Sertoli cell numbers were evaluated. Testicular cortisol concentrations were reduced at 6.5 weeks of age (P 0.05) in littermates treated with the aromatase inhibitor, letrozole, compared with littermates treated with vehicle. Letrozole treatment leads to reduced testicular estradiol and greater Sertoli cell numbers during the early juvenile interval in pigs. The inverse relationship between testicular glucocorticoid and Sertoli cell proliferation was also tested by increasing local testicular glucocorticoids using the synthetic compound, dexamethasone. Local administration beginning at 1.5 weeks of age (osmotic pump and catheter (n = 3) or a silastic implant (n = 5)) reduced Sertoli cell numbers at 6.5 weeks of age compared with littermates that received the vehicle treatment (P0.05). In summary, testicular glucocorticoid concentration was inversely correlated with Sertoli cell numbers during the first wave of Sertoli cell proliferation.

Proper, tissue-specific regulation of CYP19, the gene encoding aromatase, the key enzyme of estrogen synthesis, is essential for reproductive processes. Here, we analyzed transcriptional regulation of the porcine CYP19 in female and male gonads and brain by 5’RACE and RT-PCR and comprehensively mapped the pig CYP19 locus by in silico analysis. Our data revealed that the complete locus, including three paralogous copies, CYP19A1, CYP19A2 and CYP19A3, spans approximately 330 kb of the porcine chromosome 1. The locus also harbors the first exon of the Gliomedin gene (GLDN) in reverse orientation. Only transcripts of the CYP19A3 paralog were substantially expressed in gonads and hypothalamus. We identified CYP19A3-associated untranslated exons approximately 160 kb and 50 kb distal from the first codon. The 5´ untranslated regions of transcripts were derived from either a proximal or from one of these distal untranslated exons. Transcripts including only untranslated exons could be amplified from testis, thus suggesting long non-coding transcripts. The data revealed an additional layer of complexity in the regulation of the porcine CYP19 locus. Tissue-specific expression is not only achieved by tissue- and stage-specific expression of the three different CYP19 paralogs, but also by directing the expression of CYP19A3 from different, proximal and distal promoter regions.

Made available in DSpace on 2021-06-25T10:37:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 SUMMARY: The study reported the influence of the high and acute dose of Letrozole on the testis morphology in paca (Cuniculus paca), an aromatase inhibitor that reduces the endogenous estrogen, the essential hormone for spermatogenesis. Morphological changes were observed in seminiferous epithelium with germ cells with apoptotic characteristics and presence of vacuoles and nuclei in pycnose. Department of Surgery School of Veterinary Medicine and Animal Science University of São Paulo (FMVZ-USP), Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária Faculdade Ingá Uningá - Departamento de Medicina Veterinária, Rod PR 317, 6114-Parque Industrial Duzentos Department of Animal Morphology and Physiology University of the State of São Paulo (FCAV-UNESP), Via de acesso Prof. Paulo Donato Castellane, s/n Department of Animal Science University of California, 1 Shields Ave Department of Animal Morphology and Physiology University of the State of São Paulo (FCAV-UNESP), Via de acesso Prof. Paulo Donato Castellane, s/n

Testicular aromatase catalyzes the synthesis of estradiol, which contributes to regulation of porcine Sertoli cell proliferation and postpubertal maintenance of Sertoli cell numbers. Although aromatase enzymatic activity decreases with age and is persistently reprogrammed by prepubertal treatment with the aromatase inhibitor letrozole, the molecular bases for regulation have not been identified. DNA methylation was examined as a potential regulatory mechanism using DNA from Leydig cells isolated from 16-, 40-, and 68-week-old boars and from 68- week-old littermates treated with the aromatase inhibitor, letrozole. Methylation levels of individual CpG dinucleotides located in the distal untranslated exon 1 of the relevant aromatase encoding gene, CYP19A3, were quite high in Leydig cell DNA, and increased further with maturity of boar (P

Intranasal oxytocin (IN OXT) has been proposed as a treatment for autism spectrum disorder (ASD); however, little is known about the effects of long-term exposure. This is the first study in a non-human primate species to examine how developmental exposure to chronic IN OXT affects juvenile's interactions with family members, social preference for parents versus strangers, anxiety-like behavior, and cerebral glucose metabolism. Titi monkeys are socially monogamous and biparental; their family bonds share important characteristics with human family bonds. Fourteen males and 15 females were treated intranasally with saline (n = 14) or 0.8 IU/kg OXT (n = 15), daily from 12 to 18 months of age. Compared to SAL-treated animals, OXT-treated animals of both sexes spent significantly more time grooming other family members (F1 = 8.97, p = 0.006). Overall, OXT-treated subjects were more social (F1 = 8.35, p = 0.005) during preference tests. OXT-treated females displayed an enhanced preference for their parents (t = 2.265, p = 0.026). OXT-treated males had a blunted preference for their parents and an increase in the time spent near unfamiliar pairs (F1 = 10.89, p = 0.001). During anxiety tests, OXT-treated males refused to complete the task more often than SAL-treated males and had longer latencies (p

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Production of steroid hormones is complex and dependent upon steroidogenic enzymes, cofactors, receptors, and transporters expressed within a tissue. Collectively, these factors create an environment for tissue-specific steroid hormone profiles and potentially tissue-specific responses to drug administration. Our objective was to assess steroid production, including sulfated steroid metabolites in the boar testis, prostate, and liver following inhibition of aromatase, the enzyme that converts androgen precursors to estrogens. Boars were treated with the aromatase inhibitor, letrozole from 11 to 16 weeks of age and littermate boars received the canola oil vehicle. Steroid profiles were evaluated in testes, prostate, and livers of 16, 20, and 40 week old boars using liquid chromatography/mass spectrometry. Testis, prostate, and liver had unique steroid profiles in vehicle-treated animals. Only C18 steroid hormones were altered by treatment with the aromatase inhibitor, letrozole; no significant differences were detected in any of the C19 or C21 steroids evaluated. Testis was the only tissue with significantly decreased free estrogens following treatment with the aromatase inhibitor; estrone and estradiol concentrations were lower (p < 0.05) in testes from 16, 20, and 40 week letrozole-treated boars. However, concentrations of the sulfated conjugates, estrone-sulfate and estradiol-sulfate, were significantly decreased (p

Development of the epididymis including blood–epididymal barrier formation is not required until sperm reach the epididymis peripuberally. Regulation of this development in the early postnatal period is largely unknown. The current objectives were to evaluate potential roles of endogenous estrogen and androgen signaling during early development of the corpus epididymidis and to determine the timing of formation of the blood–epididymal barrier in the pig. Effects of endogenous steroids were evaluated using littermates treated with vehicle, an aromatase inhibitor (letrozole) to reduce endogenous estrogens, an estrogen receptor antagonist (fulvestrant) or an androgen receptor antagonist (flutamide). Phosphorylated histone 3 immunohistochemistry was used to identify proliferating epithelial cells. Lanthanum nitrate and electron microscopy were used to analyze formation of the blood barrier in the corpus epididymidis. Reducing endogenous estrogens increased the number of proliferating corpus epithelial cells at 6 and 6.5 weeks of age compared with vehicle-treated boars (PP

Previous studies have demonstrated that reducing endogenous testicular estradiol in neonatal boars would stimulate increased proliferation of Sertoli cells during the neonatal interval. The objective of this experiment was to determine if increasing testicular estradiol would have the opposite effect of reducing Sertoli cell numbers during the neonatal interval. Five littermate pairs of boars were evaluated with one littermate receiving a silastic implant containing estradiol and the second receiving only silastic at 1.5 weeks of age. Testes were recovered at 6.5 weeks of age and Sertoli cell numbers determined. Littermates treated with exogenous estradiol had approximately two-thirds as many Sertoli cells as their control littermates (P

Apical blebbing, a non-classical secretion mechanism, occurs in the mature porcine epididymis as part of its normal function. Proteins secreted by this mechanism contribute to the modification of the sperm plasma membrane during epididymal transit and are thought to contribute to acquisition of fertilizing ability. However, little is known about the regulation of this secretion mechanism in an in vivo model. Previous work demonstrated apical blebbing in the epididymis developed pubertally, suggesting androgens, sperm or other luminal factors regulated this process. Hence, the objective was to evaluate the hypothesized regulation of apical blebbing in the epididymis of pubertal boars by androgens and luminal factors. Androgen receptor blockade (flutamide) and surgical interventions (efferent duct ligation, orchidectomy or transection of the caput epididymis) were used to alter signaling, and the subsequent effects on apical blebbing were evaluated histologically. Apical blebbing was not altered by androgen receptor blockade with flutamide, but was significantly reduced 24 h after efferent duct ligation and after orchidectomy, treatments that eliminated luminal flow from the testis (P < 0.05). Like efferent duct ligation, epididymal transection altered luminal flow without removing the androgen source and significantly reduced the appearance of apical blebbing (P < 0.05). In conclusion, apical blebbing in the porcine epididymis appears to be regulated by luminal factors.

Sertoli cell numbers are a major influence on sperm production capacity. Increased use of artificial insemination and the limited number of insemination doses from a single boar ejaculate provide the incentive to increase Sertoli cell numbers for potential increases in sperm production and pig production efficiency. Although the number of Sertoli cells can be at least temporarily manipulated in vivo, the mechanism for the increased proliferation is poorly understood and critical for development of practical production practices. MicroRNAs are recognized to regulate many genes during development. Recent work suggested specific microRNAs were involved with regulation of Sertoli cell lines maintained in monoculture. In order to evaluate the relationship of specific microRNAs (miR-26a, miR-196a, miR-499, miR-638 and miR-1285) to in vivo Sertoli cell proliferation, three in vivo models of increased Sertoli cell proliferation (hemicastration, reduced androgen signaling, and reduced endogenous estrogen synthesis) were evaluated in young juvenile boars. Hemicastration was associated with slightly decreased miR-26a, similar to the decrease associated with in vitro Sertoli cell monoculture but none of the other microRNAs were affected by hemicastration nor were any affected by the blockade of androgen signaling with flutamide. Decreased expression of miR-638 followed reduced endogenous estrogen synthesis consistent with in vitro results although expression of miR-1285 was the converse from the in vitro results. Alterations in specific microRNAs are associated with increased Sertoli cell proliferation. However, altered expression of microRNAs was specific to the particular model system, suggesting unique molecular pathways are involved.

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750–1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation. J. Cell. Physiol. 230: 449–463, 2015. © 2014 Wiley Periodicals, Inc.

Sertoli cell proliferation occurs in two major waves after birth, one neonatally and another prepubertally, each contributing to final testicular size and sperm production. However, little is known about the regulation of either wave. We have previously shown that letrozole, an inhibitor of estrogen synthesis, increases Sertoli cell number and testicular size at sexual maturity in boars. These studies were conducted to determine whether letrozole affects the first or second proliferative wave. Boars were treated with letrozole during the first wave (treatment at 1, 3, and 5 weeks), less frequently (1 week of age only, or 1 and 5 weeks), on postnatal day 1, or during the second wave (weeks 11–16). Sertoli cells were enumerated in testes and estrogen concentrations were evaluated in serum and testes. Compared with vehicle controls, letrozole reduced estrogen in boars treated at weeks 1 and 5 or 1, 3, and 5, on postnatal day 1, or prepubertally. However, Sertoli cell numbers were increased only in boars treated at 1, 3, and 5 weeks of age. Neither perinatal (1 day old) nor prepubertal letrozole treatment affected Sertoli cell numbers. Hence, Sertoli cell proliferation was sensitive to letrozole only if letrozole was administered throughout the first wave, even though estrogen synthesis was effectively inhibited at all ages. These data indicate that the neonatal but not the prepubertal window of Sertoli cell proliferation is sensitive to an inhibitor of estrogen synthesis; this suggests that these two waves are differently regulated.

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm-oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm-oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona-free porcine oocytes, bound protein was visualized with 20 μg TRITC-avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration-dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm-oocyte plasma membrane interaction.

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Decreased fertility in pigs is a common occurrence during summer months. An objective of the current experiments was to evaluate if elevated ambient temperature altered the oocyte plasma membrane including potential receptors for sperm. This would potentially contribute to reduced fertilizability. Treated gilts were exposed in vivo to 32 degrees C for 12 h per day and 20 degrees C for the remaining 12 h per day for 7 days; control gilts were exposed to 22 degrees C for 12 h and 20 degrees C for the remaining 12 h each day. Cumulus-oocyte complexes were also aspirated from ovaries obtained from gilts maintained at thermoneutral ambient temperature and matured in vitro at 38.5 degrees C or 40 degrees C. Relative abundance of a porcine oocyte membrane protein was examined by intensity of immunolabelling of the in vivo and in vitro matured oocytes evaluated with confocal microscopy; fertilizability of the in vitro matured oocytes was evaluated in in vitro fertilization assays. Oocytes obtained from gilts exposed to elevated ambient temperature for 7 days had reduced immunolabelling compared with oocytes from control gilts (p < 0.05). Similarly, oocytes matured in vitro for 44 h at elevated ambient temperature had reduced immunolabelling and reduced fertilizability compared with oocytes matured at 38.5 degrees C (p < 0.01 and p < 0.05). These results suggest porcine oocyte quality is reduced by elevated ambient temperature and immunolabelling of oocytes with antibodies to specific membrane proteins may be effective to evaluate some aspects of oocyte quality.

Increased Sertoli cell proliferation during the neonatal period, transient negative effects on epididymal sperm maturation, larger postpuberal testis size reflective of increased Sertoli cell numbers, and increased testicular sperm production characterized boars subjected to continuous inhibition of endogenous estrogen production. The objective in the present experiment was to extend these previous observations to evaluate the effects of a shorter period of reduced estrogen production during the neonatal and juvenile periods on Sertoli cell proliferation, postpuberal testis size, sperm production and epididymal function. Experiments were designed to detect cumulative effects on accessory sex glands as well. Four pairs of littermate boars were assigned to the experiment with one member of each pair randomly selected to receive weekly oral treatment with the aromatase inhibitor, Letrozole, beginning at 1 week of age; the littermates received weekly oral treatment with the corn oil vehicle. Treatment stopped at 12 weeks of age and effects were examined at 10 months. Treated animals had approximately 25% larger testes (P < 0.05) correlated with increased Sertoli cell numbers (P < 0.05) and larger epididymides. Sperm quality was approximately equivalent in treated and control littermates. Accessory sex glands tended to be smaller in the treated boars. Sertoli cell proliferation during the neonatal and juvenile interval appears to be influenced by endogenous estrogen levels in the boar. A relatively short postnatal interval of Letrozole treatment effectively increased postpuberal testis size. Increased sperm production capacity in response to decreased endogenous estrogens has intriguing possibilities for animal agricultural production.

Arylsulfatase A (AS-A) is a lysosomal enzyme, which catalyzes the desulfation of certain sulfogalactolipids, including sulfogalactosylglycerolipid (SGG), a molecule implicated in cell adhesion. In this report, immunocytochemistry revealed the selective presence of AS-A in the corpus luteum of mouse ovaries. Immunoblotting indicated that mouse corpus luteum AS-A had a molecular mass of 66 kDa, similar to AS-A of other tissues. Corpus luteum AS-A was active, capable of desulfating the artificial substrate, p-nitrocatechol sulfate, at the optimum pH of five. To understand further the role of AS-A in female reproduction, levels of AS-A were determined during corpus luteum development in pseudopregnant mice and during luteolysis after cessation of pseudopregnancy. Immunocytochemistry, immunoblotting and desulfation activity showed that AS-A expression was evident at the onset of pseudopregnancy in the newly formed corpora lutea, and its level increased steadily during gland development. The increase in the expression and activity of AS-A continued throughout luteolysis after the decrease in serum progesterone levels. We also observed the selective presence of SGG on the luteal cell surface in developed corpora lutea, as shown by immunofluorescence of mouse ovary sections as well as high-performance thin-layer chromatography of lipids isolated from mouse and pig corpora lutea. The identity of the “SGG” band on the thin layer silica plate was further validated by electrospray ionization mass spectrometry. Significantly, SGG disappeared in regressing corpora lutea. Therefore, lysosomal AS-A may be involved in cell-surface remodeling during luteolysis by desulfating SGG after its endocytosis and targeting to the lysosome.

Exposure of female rats to trichloroethylene (TCE), an environmental toxicant commonly found in ground and surface waters throughout the United States, reduces the fertilizability of oocytes produced by these females compared with oocytes from control females. Localization of cytochrome P450 2E1 and glutathione s-transferase alpha, TCE-metabolizing enzymes, in the ovary suggests TCE metabolism occurs in the ovary. The production of bioactive TCE metabolites in the ovary may alter female reproductive function by altering ovarian gene transcription and/or protein expression and function. The purpose of the present study was to examine ovarian gene transcription after exposure of female rats to 0.45% TCE (v/v) in 3% Tween. Control rats received 3% Tween. Microarray analysis after 1 and 5 days of exposure indicated ovarian gene transcription was maintained during TCE exposure with the possible exception of a very few genes. Although conclusions for these few genes were ambiguous from the microarray analysis due to the minimal but statistically significant reductions, quantitative real time RT-PCR (qRT-PCR) analysis indicated expression of these genes was unaltered after TCE exposure. Protein analysis confirmed qRT-PCR results. This study suggests TCE-induced reductions in oocyte fertilizability are independent of currently detectable alterations in ovarian gene expression.

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5–4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E 2 ) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E 2 and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E 2 and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E 2 but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E 2 production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.

Reducing endogenous estrogen leads to increased proliferation of porcine Sertoli cells during the first 2 months of life. The resulting increase in porcine Sertoli cell numbers is maintained through puberty. The reduced estrogen appears to be the direct hormonal mediator because essentially no changes are observed in other hormones. However, the mechanism for this effect on Sertoli cell proliferation is unknown. The objective of these studies was to evaluate estrogen receptors alpha and beta (ESR1 and ESR2) in conjunction with androgen receptor (AR) on Sertoli cells and other testicular cell types, as an initial step toward understanding how reduced estrogen leads to increased Sertoli cell numbers. Testis sections from treated animals (aromatase inhibition to decrease endogenous estrogen beginning at 1 week of age) and from littermate controls treated with vehicle were subjected to immunocytochemical labeling for ESR1, ESR2, and AR. Three observers scored Sertoli cells, interstitial cells, peritubular myoid cells, and germ cells for intensity of labeling (0: absent; 1+: weak; 2+: moderate; or 3+: strong labeling). AR in Sertoli cells was readily detected at 1 week of age, was very faint in 2-month vehicle controls, and labeling appeared to increase in 3-month vehicle controls. AR in Sertoli cells, interstitial cells, and apparently germ cells was increased in treated animals at 2 months of age compared with the vehicle controls. This increase was confirmed in western blots. ESR1 and ESR 2 were clearly present in Sertoli cells from 1-week-old animals; ESR in Sertoli cells generally decreased with age with the decrease more apparent for ESR2. ESR1 in Sertoli cells and peritubular myoid cells exhibited some treatment-related effects but reduction of endogenous estrogen did not appear to affect ESR2 in the boar testis. The observed alterations in AR and ESR1 may mediate the increases in Sertoli cell proliferation following inhibition of endogenous estrogen production or may reflect the altered function of the Sertoli cells and peritubular myoid cells.

Aromatase cytochrome P450 (P450arom), the enzyme that catalyzes estrogen synthesis, is required for successful reproduction and is encoded by a single copy gene (CYP19) in most mammals. However, pigs and their distant suiform relatives the peccaries experienced CYP19 duplication. Here, the evolutionary origin of CYP19 duplication, and the evolution of the gene paralogs, was explored further in collared peccaries (Pecari tayassu). Exons IV and V, and the intervening intron, representing duplicated CYP19 genes, were cloned and sequenced from collared peccary, pig, and hippopotamus. Sequence alignment and analysis identified a gene conversion in collared peccary with a breakpoint 102 base pairs (bp) upstream of exon V. Phylogenetic analyses of nucleotide and amino acid sequence upstream of the breakpoint supported a tree in which one peccary sequence was orthologous with the porcine gonadal gene. Cloning and sequencing of tissue transcripts, using reverse-transcriptase polymerase chain reaction techniques (RT-PCR), confirmed that the gonadal ortholog was expressed in collared peccary testis. Orthology of the other genomic sequence with the porcine placental gene was not resolved, but its placenta-specific expression in collared peccary was confirmed by similar transcript analysis. Immunoblot and enzyme activity in collared peccary testes demonstrated much lower levels of P450arom than in pig testis. Collared peccary placental P450arom expression also seemed much lower than pigs. Thus, suiform CYP19 genes arose from an ancestral duplication that has maintained gonad- and placenta-specific expression, but at lower levels in peccaries than pigs, perhaps facilitating the emergence of different reproductive strategies as Suiformes diverged and evolved.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6–20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC–MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

Microvesicles are of increasing interest in biology as part of normal function of numerous systems; from the immune system (T cell activation) to implantation of the embryo (invasion of the trophoblasts) and sperm maturation (protein transfer in the epididymis). Yet, the mechanisms involved in the appearance of apical blebbing from healthy cells as part of their normal function remain understudied. Microvesicles are produced via one of two pathways: exocytosis or apical blebbing also termed ectocytosis. This work quantifies the histological appearance of apical blebbing in the porcine epididymis during development and examines the role of endogenous estrogens in regulating this blebbing. Apical blebbing appears at puberty and increases in a linear manner into sexual maturity suggesting that this blebbing is a mature phenotype. Endogenous estrogen levels were reduced with an aromatase inhibitor but such a reduction did not affect apical blebbing in treated animals compared with their vehicle-treated littermates. Epididymal production of apical blebs is a secretion mechanism of functionally mature principal cells regulated by factors other than estradiol.

Sertoli cell proliferation in neonatal boars is potentially androgen dependent. Hence, the immediate objective was to evaluate effects of androgen receptor-mediated signaling on the first wave of Sertoli cell proliferation. The experimental design employed littermate pairs of boars with one member assigned to receive a daily oral dose of flutamide, an androgen receptor antagonist, beginning at 1 wk of age and the littermate the canola oil vehicle. Experiment 1 examined the response at 6.5 wk of age after completion of the first wave of Sertoli cell proliferation, and experiment 2 examined the response at 11 wk of age after initiation of the second wave of Sertoli cell proliferation. Experiment 3 was designed to evaluate initial responses at 2, 3, or 4 wk of age. Additional littermates from four of the litters evaluated at 2 wk of age were hemicastrated at 8 days of age. Testis weight increased approximately 50% in the flutamide-treated boars compared with vehicle-treated littermates (P = 0.01) by 6.5 wk of age. Approximately 80% more Sertoli cells/testis were present in flutamide-treated boars at 6.5 wk of age compared with their vehicle-treated littermates (P < 0.01). Animals that were hemicastrated at 8 days of age had more Sertoli cells/testis than their intact littermates at 2 wk of age (P < 0.01), but flutamide inhibited the hemicastration response. Androgen receptor antagonism during postnatal Sertoli cell proliferation increases Sertoli cell numbers, as does hemicastration, but receptor antagonism initially inhibits Sertoli cell proliferation induced by hemicastration.

Estrogen plays an important role in male reproduction, critical for sustained fertility in some species. Reducing estrogen's interaction with its receptor(s) in monkey and mouse models is associated with reduced sperm motility and, in some cases, documented elimination of sperm fertilizing ability, suggesting that normal epididymal function may be estrogen dependent. The objective of these experiments was to evaluate the effects of reduced endogenous estrogen on development of epididymal function in the pig, a species in which males have very high levels of endogenous estrogen. Letrozole, a potent inhibitor of estrogen synthesis, was administered to neonatal boars from 1 week of age and markedly suppressed estrogen production. Epididymal function assessed as acquisition of sperm fertilizing ability (in vitro fertilization of zona-free oocytes) was reduced in Letrozole-treated animals at 24 and 28 weeks of age (23% and 30% fertilization, respectively compared with 37% and 54% in vehicle controls) but had recovered by 32 weeks of age. Cauda epididymal sperm numbers were reduced in treated animals (35% of control values at 20 weeks of age) but appeared to be recovering at 32 weeks of age. Reduction of endogenous estrogen had no effect on other aspects of epididymal function (percentage of motile sperm, sperm motion parameters, sperm head morphometrics, or ability of sperm to undergo an acrosome reaction). Reducing endogenous estrogen during postnatal development appears to have transient effects on porcine epididymal function. These transient effects suggest that the pig, with its high endogenous estrogen, may respond differently than other species to reduced estrogen synthesis.

The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

A potential endpoint for female reproductive toxicants is fertilizability of the oocytes. This endpoint has not been adequately examined for mammalian females. The objective of these studies was to evaluate fertilizability of rat oocytes following in vivo exposure to known male reproductive toxicants that exert effects via pathways that do not include endocrine disruption and to 4-vinylcyclohexene diepoxide, known to interfere with early follicular development. Oocytes were obtained from females following exposure and quality assessed by in vitro fertilization rate. One study evaluated fertilizability following 2 weeks exposure of females to inhaled tetrachloroethylene (2 h/day, 5 days/week). The remaining studies evaluated fertilizability immediately following 2 weeks exposure via drinking water to tetrachloroethylene, trichloroethylene, the fuel oxidants methyl tertiary butyl ether (MTBE), ethyl tertiary butyl ether (ETBE), tertiary amyl methyl ether (TAME), and a metabolite of the first two ethers 2-methyl-1,2-propanediol (2M2P), and to 4-vinylcyclohexene diepoxide. The percentage of oocytes fertilized was reduced following inhalation exposure to tetrachloroethylene, or consumption of trichloroethylene or TAME. Fertilizability was not altered by exposures to the other reproductive toxicants or to the other fuel oxidants. Consistent with the reduced oocyte fertilizability following exposure to trichloroethylene, oocytes from exposed females had a reduced ability to bind sperm plasma membrane proteins. Female reproductive capability assessed by the endpoint, oocyte fertilizability, was reduced by exposure to trichloroethylene and inhaled tetrachloroethylene.

Sertoli cell proliferation in neonatal boars is potentially androgen dependent. Hence, the immediate objective was to evaluate effects of androgen receptor-mediated signaling on the first wave of Sertoli cell proliferation. The experimental design employed littermate pairs of boars with one member assigned to receive a daily oral dose of flutamide, an androgen receptor antagonist, beginning at 1 wk of age and the littermate the canola oil vehicle. Experiment 1 examined the response at 6.5 wk of age after completion of the first wave of Sertoli cell proliferation, and experiment 2 examined the response at 11 wk of age after initiation of the second wave of Sertoli cell proliferation. Experiment 3 was designed to evaluate initial responses at 2, 3, or 4 wk of age. Additional littermates from four of the litters evaluated at 2 wk of age were hemicastrated at 8 days of age. Testis weight increased approximately 50% in the flutamide-treated boars compared with vehicle-treated littermates (P = 0.01) by 6.5 wk of age. Approximately 80% more Sertoli cells/testis were present in flutamide-treated boars at 6.5 wk of age compared with their vehicle-treated littermates (P0.01). Animals that were hemicastrated at 8 days of age had more Sertoli cells/testis than their intact littermates at 2 wk of age (P0.01), but flutamide inhibited the hemicastration response. Androgen receptor antagonism during postnatal Sertoli cell proliferation increases Sertoli cell numbers, as does hemicastration, but receptor antagonism initially inhibits Sertoli cell proliferation induced by hemicastration.

Both reduced endogenous estrogen and hemicastration stimulate proliferation of porcine Sertoli cells. The objective of these experiments was to compare the temporal patterns of response to each stimulus with the response to the combined stimuli as indications of shared or separate mechanisms. Within a replicate, one littermate was treated weekly with canola oil vehicle and remained intact; a second littermate was treated weekly with vehicle, and one testis was removed at Day 8; a third littermate was treated weekly with the aromatase inhibitor letrozole to reduce endogenous estrogens and remained intact; and the fourth littermate was treated weekly with letrozole, and one testis was removed at Day 8. Four replicates were evaluated at 2 wk of age, five replicates evaluated at 6.5 wk of age, and five replicates were evaluated at 11 wk of age, with treatment ceasing at 6 wk of age. Numbers of Sertoli cells were determined following GATA4 labeling using the optical dissector method. Levels of estradiol, estrogen conjugates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin were determined by radioimmunoassay. Hemicastration appeared to have a rapid effect on Sertoli cell proliferation, but letrozole treatment had no apparent effect on Sertoli cell numbers at 2 wk of age. Both letrozole treatment and hemicastration had stimulated Sertoli cell proliferation by 6.5 wk of age, although the magnitude of the hemicastration response was much greater. Letrozole appeared to have minimal interaction with hemicastration at this age. Letrozole and hemicastration together increased Sertoli cell numbers at 11 wk of age compared with either treatment alone. Estradiol and estrogen conjugates were dramatically reduced by aromatase inhibition as anticipated; treatment-induced changes in inhibin, LH, or FSH were minimal. Differences in timing of response and positive interaction at 11 wk of age suggest that hemicastration and letrozole stimulate proliferation of Sertoli cells by two initially different pathways.

The percentage of offspring sired after heterospermic insemination of equal numbers of spermatozoa is believed to be a very sensitive measure of relative in vivo fertility of the inseminated samples. The objective of these trials was to evaluate whether there was a detectable male-female interaction in the fertilizing ability of spermatozoa. If there was such an interaction, we reasoned that the paternity of offspring from individual females in a heterospermic trial the second year would be similar to the paternity of offspring in the same individual females the first year if the same ejaculates were used. Five groups of ewes were inseminated with different combinations of semen (a single Merino ejaculate from one of five rams randomly paired with five different pools of Suffolk semen) in a heterospermic trial. Those ewes conceiving the first year were inseminated in a second breeding season with the same combination of semen used previously. The percentage of lambs sired by each ejaculate/pool of ejaculates was calculated for all lambs born from all ewes inseminated with each semen combination. These percentages would be the expected ratios of Merino-sired:Suffolk-sired lambs if there is no male-female interaction. Ewes in each group were divided into two subgroups: those conceiving only Merino-sired lambs the first year and those conceiving at least one Suffolk-sired lamb the first year. The ratio of Merino-sired lambs:Suffolk-sired lambs did not differ in either subgroup from those expected if there was no male-female interaction. These results are consistent with the absence of a male-female interaction in relative fertilizing ability of spermatozoa.

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.

The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.

In the present study, α-chlorohydrin (ACH) (5, 10, 25, 50 and 75 mg/kg, po) was administered to rats and the effects on sperm ATP levels, sperm motility, and the ability of sperm to bind and penetrate rat oocytes were determined. Groups of rats were killed 5 days and 3 h following treatment. At both time points, sperm from ACH-treated rats (≥10 mg/kg) had significantly lower levels of ATP when diluted in media containing glucose. No diminution of ATP was seen in sperm diluted in phosphate-buffered saline (PBS). Computer analysis of sperm motility indicated that straight-line velocity (VSL) was the most sensitive parameter to ACH treatment and was significantly decreased in rat sperm three hours after ACH exposure (25 mg/kg). A clear drop in percent penetration (35% vs. 85% in control) of zona-free rat oocytes by rat sperm of both ACH groups was observed at 10 mg/kg. Higher dose levels produced no significant further decrease in percent penetration. Overall, the fertilizing ability of sperm was highly sensitive to ACH doses that caused minor but significant changes in sperm ATP levels and no significant changes in motility. These data are consistent with the spermatozoan’s need for an uncompromised energy supply to maintain its ability to bind and penetrate the oocyte.