Effect Of Spermatozoa Research Articles

The Effects of Sperm and Seminal Fluid of Immunized Male Mice on In Vitro Fertilization and Surrogate Mother–Embryo Interaction

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination’s effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C–C, Im–C, C–Im, and Im–Im. The pre-implantation losses were significantly lower in the Im–C group than in the C–Im group. At the same time, the resorption rates reduced markedly in the C–Im compared to the Im–C group. Embryo and placenta weights were significantly higher in the Im–Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im–Im group, they were strongly correlated with embryo mass. The number–size trade-off was only significant in the Im–Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males’ antigenic challenge on the IVF success rate.

Human sperm DNA damage has an effect on immunological interaction between spermatozoa and fallopian tube.

Toll-like receptors play a crucial role in the immunological interaction between the spermatozoa and fallopian tube and contribute to the ovulation, sperm capacitation, fertilization, and pregnancy. To investigate the expression of toll-like receptors and their adaptor molecules and cytokines under the effect of spermatozoa with high DNA fragmentation (high DF) in human fallopian tube cell line (OE-E6/E7) and compare to those in normal spermatozoa. Fresh semen samples were obtained from 10 unexplained infertile males with high DF (more than 20%) and from 10 healthy donors with a DF less than 3%. After sperm preparation, samples were co-cultured with OE-E6/E7. Toll-like receptors, myeloid differentiation factor 88 (MyD88), TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing IFN-b (TRIF), TRIF-related adapter molecule as well as IL-6, IL-8, IFN-β, and TNFα mRNA expression were evaluated by quantitative real-time PCR. Protein levels of these cytokines and chemokines were measured using ELISA method. TLR 1-6 mRNA expression in OE-E6/E7 was significantly higher under the effect of spermatozoa with high DF compared to the spermatozoa with low DF. Furthermore, significantly increased mRNA expression of MyD88, TIRAP, and TRIF was observed in the high DF group compared to the low DF group, except TRIF-related adapter molecule. Moreover, the expression of IL-6 and IL-8 in the high DF group was significantly higher than low DF group, although there was no significant difference in IFN-β and TNFα expression between the groups. Damage-associated molecular patterns from DNA damage activate TLR signaling pathway in human fallopian tubes and result in the upregulation of inflammatory cytokines and chemokines. This situation may provide pathologic environment for capacitation, fertilization, embryo development, and implantation in female reproductive tract and can be one of the mechanisms of infertility in men with high DF.

69 Can Bovine Sperm Interaction with the Oviduct Cells After Artificial Insemination Affect the Transcriptome Profile of the Oviduct?

During their transit through the female genital tract, spermatozoa bind to oviduct cells, where they are maintained alive for a long period. Although some studies have been carried out to evaluate the effects of the oviduct on spermatozoa, less is known about the effect of spermatozoa on the oviduct. The aim of this study was to evaluate the effect of sperm on the transcriptome profile of the bovine oviduct after fixed-time AI (FTAI). Nellore cows submitted to FTAI were randomly inseminated using 8 × 106 frozen–thawed spermatozoa (sperm group, n = 9) or saline solution (control group, n = 9). Eighteen hours after FTAI, the cows were slaughtered, and the cells from oviducal isthmus were collected and stored in liquid nitrogen until transcriptome analysis. For RNA extraction, 3 pools of 3 oviducts/cows from each group were formed. Total RNA was extracted and the RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcription profiling from each group was performed by HiSEqn 2500 (Illumina Inc., San Diego, CA, USA). After sequencing, the data were analysed by edgeR software, and differential gene expression analysis between sperm and control group was performed using the generalized linear model (P = 0.05). The P-value was adjusted by false discovery rate (FDR) Benjamini-Hochberg method, to avoid false positives. Moreover, using mRNA expression levels (log fold change and P-values) as coefficients, functional enrichment analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. A total of 9,374 genes were found to be expressed in bovine oviducal isthmus, of which 219 genes were differentially expressed between sperm and control group. Differentially expressed genes genes were classified according to 3 major GO classifications, with 10 gene categories in molecular function, 3 gene categories in cellular component, and zero categories in biological process. In the molecular function category, genes related to calcium ion binding (n = 113), molecular transducer activity (n = 111), and receptor activity (n = 111), were predominant. For cellular component, the highest number of genes (n = 176) was related to extracellular space. Moreover, KEGG assignments were used to classify the functional annotations of the pathways genes to further understand the biological functions of the expressed genes. It was shown that steroid hormone biosynthesis, arachidonic acid metabolism, calcium signalling, cytokine-cytokine receptor interaction, neuroactive ligant-receptor interaction, extracellular matrix-receptor interaction, cell adhesion molecules, coagulation and complement cascades, ovarian steroidogenesis, protein digestion and absorption and mineral absorption pathways had genes with higher or lower expression than the median of gene expression out of the tested category. The results suggested that bovine sperm affect the transcriptome profile of the bovine oviduct after FTAI. Moreover, based on the functional enrichment analysis, an effect of the sperm-oviduct interaction on some pathways analysed by GO and KEGG was identified. This research was financially supported by FAP-DF, FAPESP, CNPq.

No cytotoxic effects from application of pentoxifylline to spermatozoa on subsequent pre-implantation embryo development in mice

Abstract The aim was to assess the effect of spermatozoa exposed to PTX on the rates of fertilization and embryo development and apoptotic cells within blastocysts in an animal model. Mice Oocytes were inseminated with spermatozoa exposed to 3.6 mmol PTX for 30 min, or with neat spermatozoa. Then fertilization and embryo development rate, blastocyst formation and quality, as well as total cell number of blastocyst, and DNA fragmentation index (DFI) in blastocysts were surveyed in both groups. Fertilization and embryo development rate were similar between the groups. The rates of blastocyst formation did not differ significantly between control and PTX groups (52.4% vs. 51.8%). The average of total cell count in blastocysts and DFI in control and PTX groups were also insignificant (31.08 ± 1.5 vs. 34.14 ± 1.5 and 9.76 ± 5.0 vs. 11.77 ± 5.4). Application of PTX for enhancing sperm motility does not cause a cytotoxic effect on subsequent embryo development and embryo genome integrity.

A novel approach to assessing bisphenol-A hazards using an in vitro model system.

BackgroundAlthough the toxicological impacts of the xenoestrogen bisphenol-A (BPA) have been studied extensively, but the mechanism of action is poorly understood. Eventually, no standard method exists for evaluating the possible health hazards of BPA exposure. Considering mice spermatozoa as a potential in vitro model, we investigated the effects of BPA exposure (0.0001, 0.01, 1, and 100 μM for 6 h) on spermatozoa and the related mechanisms of action. The same doses were also employed to evaluate protein profiles of spermatozoa as a means to monitor their functional affiliation to diseases.ResultsOur results demonstrated that high concentrations of BPA negatively affect sperm motility, viability, mitochondrial functions, and intracellular ATP levels by activating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A pathways. Moreover, short-term exposure of spermatozoa to high concentrations of BPA induced differential expressions of 24 proteins. These effects appeared to be caused by protein degradation and phosphorylation in spermatozoa. Proteins differentially expressed in spermatozoa from BPA treatment groups are putatively involved in the pathogenesis of several diseases, mainly cancer, carcinoma, neoplasm, and infertility.ConclusionsBased on these results, we propose that BPA adversely affects sperm function by the activation of several kinase pathways in spermatozoa. In addition, BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa, subsequently involve in the pathogenesis of many diseases. Therefore, we anticipated that current strategy might broadly consider for the health hazards assessment of other toxicological agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2979-5) contains supplementary material, which is available to authorized users.

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice.

Background:Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood.Objectives:The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice.Methods:Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease.Results:BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases.Conclusions:Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa.Citation:Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238–245; http://dx.doi.org/10.1289/EHP378

Water contaminated with Didymosphenia geminata generates changes in Salmo salar spermatozoa activation times

Didimosphenia geminata (“didymo”), has become a powerful and devastating river plague in Chile. A system was developed in D. geminata channels with the purpose evaluating the effects of water polluted with didymo on the activation of Atlantic salmon (Salmo salar) spermatozoa. Results indicate that semen, when activated with uncontaminated river water had an average time of 60±21s. When using Powermilt, (a commercial activator), times of 240±21s are achieved, while rivers contaminated with D. geminata achieve a motility time of 30±12s. Interestingly enough, the kinetic parameters of VSL, VCL and VAP showed no significant changes under all of the conditions. Furthermore, the presence of D. geminata reduces activation time of the samples as the cells age, indicating increased effects in spermatozoa that are conserved for more than 5 days. D. geminata has antioxidant content, represented by polyphenols; 200ppm of polyphenol were obtained in this study per 10g of microalgae. Spermatozoa exposed to these extracts showed a reduction in mobility time in a dose dependent manner, showing an IC50 of 15ppm. The results suggest an effect on spermatozoa activation, possibly due to the release of polyphenols present in contaminated rivers, facilitating the alteration of sperm motility times, without affecting the viability or kinetics of the cells. These findings have important implications for current policy regarding the control of the algae. Current control measures focus on the number of visible species, and not on the compounds that they release, which this study shows, also have a problematic effect on salmon production.

Evaluation of immunological interaction between spermatozoa and fallopian tube epithelial cells.

Toll-like receptors (TLR) are one of the major compartments of innate immune system. It was revealed that the TLR have relevance in ovulation, sperm capacitation and fertilisation. So, in this study, the expression of TLR, their adaptor molecules and cytokines in human fallopian tube cell line under the effect of human normal spermatozoa was evaluated. TLR mRNA and protein were evaluated in OE-E6/E7 cell line. Semen samples from 10 donors were collected and co-incubated with OE-E6/E7 cell line and used as sperm group, and cell line without spermatozoa was used as control group. Afterwards, the level of TLR, their adaptor molecule and cytokine mRNA expression was compared using qPCR in sperm and control groups, and supernatant was used for ELISA. To determine whether elevated cytokine reaction to spermatozoa in OE-E6/E7 cell line is mediated via TLR, TLR3 function-blocking antibody was used. OE-E6/E7 cell line expressed TLR1-6 genes and proteins. TLR expressions, especially TLR3 and TLR5, in OE-E6/E7 cell line under the effect of spermatozoa were significantly higher. Also, levels of adaptor molecules and cytokine production were increased in sperm group than in control group (P < 0.05). So, it may be hypothesised that TLR are essential for spermatozoa and fallopian tube immunological interaction and for preparing safe environment for important events in fallopian tube.

Immunobiology of Spermatozoa: A Review

Immunobiological changes in spermatozoa within the male reproductive tract are vital for their viability, and hence for reproductive efficiency. The secretions of the seminal vesicles have immunosuppressive property that attenuates the antigenic effects of spermatozoa in the male reproductive tract. However, when spermatozoa find a way to outside of the rete testis or epididymis due to trauma or infection, they can cause significant inflammatory and immunological reactions in the surrounding tissues. This has also been observed in the female reproductive tract. However, such cases are rare and the reason for this is unknown. Antisperm antibodies are the main cause of infertility in humans. The purpose of this review is to analyze these immunobiological changes from the time of spermatozoa formation in rete testis to their role in fertilizing the oocytes in the female reproductive tract.

The Effect of the Separation of Spermatozoa X and Y Using Swim up Method on the Quality of Etawah Crossbreed Goats Spermatozoa

This research aimed to determine the effect of spermatozoa X and Y separation using swim-up method on the quality of etawah crossbreed goat’s spermatozoa. This research used 6 male etawah crossbreed goats aged between 18-24 months, which were collected the semen once a week using electroejaculator. Immediately after semen collection, the quality of semen was examined, and then grouped into three treatment groups. Group 1 was control group (P0) refers to the semen was not separated by swim-up method. Group 2 (P1) was the semen separated by the swim-up method for 5 minutes. Group 3 (P2) was the semen separated by swim-up method for 10 minutes. Each treatment was repeated six times. Parameters for semen quality examined were the percentage of spermatozoa motility, live spermatozoa, and abnormal spermatozoa. Data were analyzed by analysis of variance (ANOVA) one way pattern, followed by Duncan's multiple test. The results showed that the mean ± SD percentage of sperm motility on each treatment groups (P0; P1; P2) was 81.33±3.44, 89.67±3.21, and 90.00±3.10 %, respectively. The percentage of live spermatozoa on each group was 86.50 ± 2.07, 92.33 ± 2.08, and 91.83 ± 1.72%, respectively. The percentage of abnormal spermatozoa on each group was 13.00±2.53, 7.33±2.52, and 7.67±2.16%, respectively. Results of this research showed that the percentages of motility, live spermatozoa, and abnormal spermatozoa after separation by swim-up method were significantly different (P0.05) compared to without separation. It can be concluded that the separation of spermatozoa using swim-up method can improve significantly (P0.05) the percentage of live and motility of spermatozoa and decrease the percentage of abnormal spermatozoa of etawah crossbreed goats.Key words: Etawah crossbreed, spermatozoa quality, swim-up method

AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility.

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca2+ and bicarbonate TCM, time from 1–24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 µM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca2+ and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.

Ca2+, Mg2+-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin Strongylocentrotus intermedius Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca(2+), Mg(2+)-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn(2+) blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

Effects of spermatozoa during in vitro meiosis progression in the porcine germinal vesicle oocytes

We have investigated the effect of co-culture with porcine spermatozoa on in vitro maturation of porcine germinal vesicle (GV) oocytes before fertilization. Most oocytes were arrested at the first prophase of meiosis when oocytes were cultured in TCM 199 alone, but the proportion of oocytes that reached metaphase II was significantly elevated by co-incubation with spermatozoa in vitro. The oocyte maturation effect was observed with intact and parts of spermatozoa (head and tail) collected from adult swine (regardless of source). However, gonocytes from the newborn porcine testis were not able to enhance in vitro maturation of porcine germinal vesicle oocytes. Interestingly, the oocyte maturation effect by spermatozoa was not decreased with heat treatment, but the maturation effect of oocyte treatment disappeared with exposure to detergent in sperm suspension. Porcine spermatozoa were also observed to stimulate meiosis of oocytes, which was maintained at meiotic arrest using dibutyryl cyclic AMP or forskolin. The study suggests that (i) membrane of porcine spermatozoa contains a substance(s) that can enhance in vitro maturation of oocytes prior to fertilization, (ii) the putative meiosis-enhancing substance(s) of spermatozoa from adult testes retains the oocyte maturation effect during transportation of spermatozoa through epididymis, and (iii) the putative meiosis-enhancing substance(s) is able to overcome the inhibitory effect of dibutyryl cyclic AMP or forskolin by inducing germinal vesicle breakdown of porcine cumulus–oocyte complexes maintained in meiotic arrest.

Changes in intracellular distribution and activity of protein phosphatase PP1gamma2 and its regulating proteins in spermatozoa lacking AKAP4.

The second messenger cAMP mediates its intracellular effects in spermatozoa through cAMP-dependent kinase (PKA, formally known as PRKACA). The intracellular organization of PKA in spermatozoa is controlled through its association with A-kinase-anchoring proteins (AKAPs). AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is sperm specific and the major fibrous sheath protein of the principal piece of the sperm flagellum. Presumably, AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function. It is also proposed to act as a scaffolding protein for signaling proteins and proteins involved in metabolism. Akap4 gene knockout mice are infertile due to the lack of sperm motility. The fibrous sheath is disrupted in spermatozoa from mutant mice. In this article, we used Akap4 gene knockout mice to study the effect of fibrous sheath disruption on the presence, subcellular distribution, and/or activity changes of PKA catalytic and regulatory subunits, sperm flagellum proteins PP1gamma2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC), GSK-3 (glycogen synthase kinase-3), SP17 (sperm autoantigenic protein 17, formally known as SPA17), and other signaling proteins. There were no changes in the presence and subcellular distribution for PP1gamma2, GSK-3, hsp90 (heat shock protein 1, alpha, formally known as HSPCA), sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7), 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), and PKB (thymoma viral proto-oncogene, also known as AKT) in mutant mice. However, the subcellular distributions for PKA catalytic subunit and regulatory subunits, PI 3-kinase (phosphatidylinositol 3-kinase), and SP17 were disrupted in mutant mice. Furthermore, there was a significant change in the activity and phosphorylation of PP1gamma2 in mutant compared with wild-type spermatozoa. These studies have identified potentially significant new roles for the fibrous sheath in regulating the activity and function of key signaling enzymes.

Effects of heparin, sperm concentration and bull variation on in vitro fertilization of bovine oocytes in a protein-free medium

The present study was conducted to examine the effects of heparin, sperm concentration and bull variation on the fertilization of bovine oocytes in a protein-free medium supplemented with polyvinyl alcohol and subsequent in vitro development of fertilized embryos. The effects in protein-free medium were compared with those in medium supplemented with bovine serum albumin (BSA). In the presence of heparin (1, 10 and 100 μg/ml), nearly all the oocytes were fertilized with and without BSA. In the absence of BSA, polyspermy was lower (4 to 15%) than in its presence (15 to 48%; P < 0.05). An increase in sperm concentration from 1 × 10 4 cells/ml during insemination enhanced fertilization rate up to 1 × 10 6 cells/ml with and without BSA (14 to 90% and 3 to 77%, respectively). In the absence of BSA, the highest concentration of spermatozoa (1 × 10 7 cells/ml) gave a lower fertilization rate (55%) than that at 1 × 10 6 cells/ml (77%; P < 0.05). Polyspermy neither increased nor decreased sperm concentration without BSA (0 to 8%; P > 0.05). The effects of spermatozoa from 5 different bulls chosen randomly on in vitro fertilization in medium without BSA were examined. Individual bull variation in fertilization rate (36 to 95%) was noted at 3 different heparin concentrations (1, 10 and 100 μg/ml). Polyspermic fertilization was low (0 to 14%) and was the same for all bulls at all heparin concentrations. Embryos fertilized without BSA developed to the blastocyst stage at the same rate (27%) as those with BSA (33%; P > 0.05).

Rabbit sperm function after co-culture with uterine epithelial cells

The effects of spermatozoa:uterine epithelial cell interactions in vitro on various sperm functions were studied using monolayers of uterine epithelial cells, endometrial stromal cells and fetal fibroblasts. Epithelial and stromal cells were isolated from uteri of rabbits in estrus, while fibroblasts were derived from 12-d-old rabbit fetuses. Twenty-nine to 31 h after culture initiation, washed, ejaculated rabbit spermatozoa were incubated with epithelial cells, uterine stromal or fetal fibroblastic cells, medium or conditioned medium. Sperm viability and loss of acrosome were measured after 10 to 20 h of incubation. Progressive sperm motility and fertilizing ability, which was assessed by an in vivo fertilization assay, were determined after 12 h co-culture. Sperm viability decreased throughout the culture period and was not affected by treatment. Sperm co-cultured with epithelial cells or incubated in medium had fewer acrosomes after 20 h than after 10 h. Fewer sperm co-cultured with stromal or fibroblastic cells lost their acrosomes. Progressive motility was positively affected by sperm-epithelium interaction as 39% of the co-cultured sperm were motile compared with 18% of the sperm incubated in media. In vivo fertilization experiments suggested that sperm incubated with epithelial cells or in medium had similar fertilizing ability. The co-culture of sperm with uterine cells provides an in vitro model to evaluate the effect of gamete-genital tract interaction on sperm function.

Re-evaluation of the role of spermatozoa as inducers of protein synthesis by the rabbit endometrium.

The effect of spermatozoa as inducers of protein synthesis by the rabbit endometrium was studied. The presence of spermatozoa increased the leucine incorporation into proteins from 15.6 +/- 1.8 to 44.8 +/- 4.3 dpm X 10(-3)/mg DNA after 2 hr of incubation. There was no difference in the amount of incorporated leucine induced by live or by dead spermatozoa. Latex particles also induced an increase on protein synthesis (24 +/- 3.1 dpm X 10(-3)/mg DNA). These results seem to indicate that this increase in protein synthesis was nonspecific. The number of contaminant cells was always significantly greater in the recovered incubation medium after incubation with highly metabolically active sperm than with dead cells (230 +/- 85 and 86 +/- 33 X 10(-3) cells respectively). This leukocytic effect was smaller than that found in In vivo systems and may play a significant role on the proposed activity of sperm cells on the endometrium.

The stage of spermatogenesis involved in the testicular-hypophyseal feed-back mechanism in man.

ABSTRACT In 284 patients with a great variety of primary testicular disorders registration of spermatogenesis was performed with the testicular biopsy score count method described by the author. Correlation analyses between the score count steps and gonadotrophin excretion show a great rise when spermatozoa (late spermatids) disappear from the testis. There is a high correlation between the per cent of tubuli possessing spermatozoa and the gonadotrophin level. Disappearance also of spermatids or spermatocytes or spermatogonia induces no further rise in gonadotrophins and no separate effect of these cells on gonadotrophins could be demonstrated. Some influence upon the gonadotrophin level was found when only Sertoli cells were present but this was small as compared with the effect of spermatozoa. It is concluded that the stage of spermatogenesis involved in the testicularhypophyseal feed-back mechanism in man is the last stage where the spermatozoon is finished and matured. The way this stage exerts its influence upon the gonadotrophin level is briefly discussed.