Comparison of Botu-bov and Tris as Freezing Extenders of Buffalo Sperm Recovered from Epididymal Cauda

Valuable genetic material can be preserved by the cryopreservation of epididymal sperm. This study evaluated the viability of pre-freezing and post-thawed sperm samples recovered from the epididymal cauda of buffaloes. Epididymides from eight Murrah buffaloes with 18 months of age were used. Semen samples were diluted in two different freezing extenders: Botu-bov (BB) and Tris (TRIS). Immediately after slaughter, both testicles from each animal were collected and transported at 4 degrees C for six hours interval. In laboratory, the removed epididymides were flushed to obtain sperm and the fractions were diluted in both freezing extenders (BB and TRIS). Semen doses were analyzed before and after frozen at -196 degrees C. BB and TRIS pre-freezing results were 38.54 +/- 22.33%(b) and 14.17 +/- 12.78%(a) for total motility (TM), 25.00 +/- 16.12(a) and 9.44 +/- 9.11(a) for progressive motility (PM), 7.21 +/- 0.98(a) and 5.09 +/- 2.65(a) for percentage of rapid cells (RAP), 91.08 +/- 12.53(b) and 63.33 +/- 31.47(a) for velocity of trajectory (VAP), 73.54 +/- 20.17(b) and 49.50 +/- 9.11(a) for linear progressive velocity (VSL), 172.21 +/- 24.55(a) and 116.94 +/- 59.48(a) for curvilinear velocity (VCL), respectively (P < 0.05). BB and TRIS post-thawing results were 42.25 +/- 21.50(b) and 17.62 +/- 19.46(a) for TM, 27.25 +/- 24.86(a) and 18.00 +/- 13.68(a) for PM, 7.35 +/- 0.98(a) and 6.26 +/- 1.13(a) for RAP, 91.42 +/- 16.86(a) and 75.96 +/- 13.17(a) for VAP, 67.96 +/- 12.13(a) and 60.04 +/- 10.42(a) for VSL, 177.54 +/- 23.53(b) and 141.29 +/- 24.97a for VCL, respectively (P < 0.05). The sperm recovered from the epididymal cauda, after 6 h storage of epididymides at 5 degrees C ensures sperm preservation demonstrating that the diluent Botu-bov had higher total motility both pre-and post-freezing when compared with TRIS. Additionally, the sperm frozen with the diluent Botu-bov showed higher values of VSL at post-thawing. These findings may reflect in improvement of conception rates.

Influence of sperm motility factors on spermatozoa obtained from diffent epididymal segments

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructure of the epithelium lining of cauda epididymidis in mongrel dogs

The epithelium lining of cauda epididymidis in mongrel dogs was examined by transmission electron microscopy. The epididymal epithelium is pseudostratified with stereocilia and is composed predominantly of principal and clear cells. Therefore, exist basal and apical cells. The principal and clear cells show features suggesting that they may be preferentially involved in absorptive and secretive functions. These results are compared with previously published data on the cauda epididymidis of other mammalian species, in order to understand the significance of the epididymis in sperm maturation.

Novel genes and regulatory systems in epididymal differentiation and sperm maturation of the mouse.

Mammalian spermatozoa gain their fertilizing ability during maturation in the epididymis. Proteins and lipids secreted into the epididymal lumen remodel the sperm membrane, thereby providing the structure necessary for progressive motility and oocyte interaction. In the current study, genetically modified mouse models were utilized to determine the role of novel genes and regulatory systems in the postnatal development and function of the epididymis. Ablation of the mouse β-defensin, Defb41, altered the flagellar movements of sperm and reduced the ability of sperm to bind to the oocyte in vitro. The Defb41-deficient iCre knock-in mouse model was furthermore utilized to generate Dicer1 conditional knock-out (cKO) mice. DICER1 is required for production of mature microRNAs in the regulation of gene expression by RNA interference. Dicer1 cKO gave rise to dedifferentiation of the epididymal epithelium and an altered expression of genes involved in lipid synthesis. As a consequence, the cholesterol:polyunsaturated fatty acid ratio of the Dicer1 cKO sperm membrane was increased, which resulted in membrane instability and infertility. In conclusion, the results of the Defb41 study further support the important role of β-defensin family members in sperm maturation. The regulatory role of Dicer1 was also shown to be required for epididymal development. In addition, the study is the first to show a clear connection between lipid homeostasis in the epididymis and sperm membrane integrity. Taken together, the results give important new evidence on the regulatory system guiding epididymal development and function

Untersuchungen zur Eliminierung und Resorption von Zink aus Säugerspermatozoen während der Nebenhodenpassage

Während der Spermatogenese wird das Element Zink an die Sulfhydrylreste der Cysteine in den Mantelfaserproteinen der Spermienflagellen gebunden. So kann in den noch unreifen Mantelfasern die ungerichtete Ausbildung von Disulfidbrücken verhindert werden. Im Zuge der Spermatozoenreifung während der Nebenhodenpassage wird dieses Zink androgenabhängig zu einem hohen Prozentsatz wieder eliminiert. Die nun gerichtete Ausbildung von Disulfidbrücken ermöglicht die Versteifung der Mantelfasern. Diese Rigidität stellt die Voraussetzung zur progressiven Motilität dar, ohne die eine Fertilisierung der Eizelle im weiblichen Genitaltrakt nicht möglich ist. Da eine negative Korrelation zwischen dem Zinkgehalt von Flagellen und ihrer Motilität besteht (Henkel et al., 1999), hat die Zinkeliminierung während der Nebenhodenpassage eine entscheidende Bedeutung in der Entwicklung der Spermatozoen. Die vorliegende Arbeit untersucht die Mechanismen der epididymalen Zinkeliminierung sowie die an diesem Prozess beteiligten Komponenten und den Verbleib des eliminierten Zinks am System des Bullen, der Ratte und des Menschen. Mittels proteinchemischer Verfahren kann im bovinen System ein zinkbindendes 60 kDa-Protein als Albumin identifiziert werden. Ein 80 kDa-Protein mit zinkbindenden Eigenschaften bleibt unidentifiziert. Die Zinkbindungskapazität der fraktionierten Proteine ist dabei im Caput epididymidis am stärksten ausgeprägt. Atomabsorptionsspektralphotometrische Untersuchungen zeigen die höchsten flagellären Zinkwerte in den Spermien des Rete testis und eine Abnahme des Zinkgehalts zwischen Nebenhodenkopf und -körper. Durch Autometallographie kann eine epitheliale Zinkresorption im Nebenhodenschwanz nachgewiesen werden. Dort erfolgt auch die basale Anreicherung des Zinks. Am Zinkstoffwechsel scheint auch der Macrophage Migration Inhibitory Factor (MIF) beteiligt zu sein. Dieser ist ein Zytokin mit Oxidoreduktasecharakter und kommt unter anderem im Nebenhodenepithel sowie in den Vesikeln des Nebenhoden-Fluids der Ratte vor. MIF zeigt in den hier durchgeführten Untersuchungen sowohl in systemhomologer, als auch in rekombinanter Form in vitro eine Zink-eliminierende Wirkung auf Rattenspermatozoen des Caputs und der Cauda epididymidis und hat somit möglicherweise Einfluss auf den Reifungsprozess der Spermien im Nebenhoden. Lit.: Henkel R, Bittner J, Weber R, Hüther F, Miska W (1999). Relevance of zinc in human sperm flagella and its relation to motility. Fertil Steril 71: 1138-1143

Anàlisi microscòpica de l'esperma ejaculada i de la maduració epididimària dels espermatozoides de Sus domesticus

El present treball analitza la morfologia espermàtica de l'ejaculat de Sus domesticus, la histologia del conducte epididimari i la qualitat de l'esperma epididimari. El material d'estudi prové de mascles reproductors porcins de les races Landrace i Pietrain, sans i sexualment madurs. La metodologia emprada es basa en l'examen al microscopi òptic (camp dar, contrast de fases i contrast interferencial) i al microscopi electrònic (de rastreig i de transmissió). Per a l'anàlisi estadística de les dades s'ha utilitzat el test de la X2 de Pearson (p<0,01). L'estudi de la morfologia espermàtica de l'ejaculat permet distingir diversos tipus de gàmetes que s'han classificat en tres grups: espermatozoides madurs, espermatozoides immadurs i espermatozoides aberrants, així com algunes cèI.lules somàtiques. L'espermatozoide madur de Sus domesticus és un gàmeta típic de mamífer (format per tres parts: cap, peça de connexió i cua) en que destaquen: la forma oval i plana del cap, el desenvolupament d'una protuberància acrosòmica apical en una de les cares del cap i la presencia dels cossos laminars en la peça de connexió. L'espermatozoide immadur es caracteritza per la presencia de la gota citoplasmàtica, el major desenvolupament de la protuberància acrosòmica apical i per la flexibilitat del cap. Els espermatozoides aberrants es descriuen i classifiquen segons la morfologia externa i la morfologia interna, distingint-se una amplia gama de malformacions que afecten les diverses parts de l'espermatozoide. Les cèl·lules somàtiques presents en l'ejaculat ofereixen les característiques pròpies d'un macròfag i se les ha observat englobant espermatozoides immadurs. L'estudi de l'estructura i la ultraestructura de les tres regions anatòmiques de l'epidídim (caput, corpus i cauda) revela que: a) l'epiteli epididimari és pseudoestratificat amb esterocilis, b) cada regió epididimària presenta uns valors característics en relació al diàmetre intern del conducte, a l'alçada de l'epiteli, a la longitud dels esterocilis i al nombre de cèl·lules somàtiques luminals, i c) l'epiteli epididimari esta format per cinc tipus cel·lulars: les cèl·lules principals, les cèl·lules basals, les cèl·lules dares, les cèl·lules estretes i les cèl·lules basòfiles. Dels resultats obtinguts es pot deduir que: a) aquests cinc tipus cel·lulars es distribueixen al llarg del conducte epididimari de forma no homogènia, b) les cèl·lules basals, les cèl·lules principals, les cèI.Iules dares i les cèl·lules estretes són diversos estadis del desenvolupament d'un mateix tipus cel·lular especialitzat en la secreció i reabsorció cel·lular, i c) les cèl·lules basòfiles són les precursores de les cèl·lules somàtiques luminals. La qualitat de l'esperma procedent de les tres regions de l'epidídim ha estat analitzada a partir dels següents paràmetres espermàtics: vitalitat, resistència osmòtica dels acrosomes, estabilitat cefàlica, morfologia, malformacions i aglutinació. La vitalitat espermàtica disminueix progressivament al llarg del conducte epididimari. La resistència osmòtica dels acrosomes s'assoleix en la regió corporal de l'epidídim. L'estabilitat cefàlica dels espermatozoides és més elevada en les dues primeres regions de l'epidídim que en la regió caudal. Cada regió de l'epidídim es caracteritza per una morfologia espermàtica específica: a) el caput es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica proximal, b) el corpus es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica distal, i c) el cauda es caracteritza per l'elevat percentatge d'espermatozoides madurs. S'han estudiat les següents malformacions d'origen epididimari: espermatozoides de cua doblegada per l'anell de Jensen (origen en el cauda), espermatozoides de cua enrotllada i espermatozoides de cues fusionades (origen en el corpus). Els espermatozoides perden la capacitat de doblegar la cua per la peça intermèdia a mesura que avancen pel conducte epididimari. L'aglutinació espermàtica tendeix a augmentar progressivament al llarg del conducte epididimari, si bé, no s'han observat variacions significatives en els diversos tipus d'aglutinació. La maduració epididimaria dels espermatozoides de Sus domesticus és un procés lent i complex, i la qualitat de l'ejaculat depèn de que aquesta maduració hagi estat completa. La presencia en l'esperma ejaculat de formes gamètiques pròpies de l'esperma epididimari és un signe d'una incompleta maduració dels espermatozoides; i, pot considerar-se com un paràmetre indicador d'estrés del mascle reproductor, tant més quant més s'assembli a la morfologia espermàtica de la regió cefàlica de l'epidídim.

Artificial oocyte activation with calcium ionophore A23187 in intracytoplasmic sperm injection cycles using surgically retrieved spermatozoa

Objective: To evaluate the effect of artificial oocyte activation (AOA) on intracytoplasmic sperm injection (ICSI) cycles using surgically retrieved sperm.Design: Laboratory study.Setting: Fertility/assisted fertilization center.Patient(s): Couples undergoing surgical sperm retrieval for ICSI (n = 204).Intervention(s): Application of calcium ionophore A23187 for AOA.Main Outcome Measure(s): Cycles were divided into experimental groups according to the origin of the sperm used for injection and the type of azoospermia: [1] testicular sperm aspiration in nonobstructive-azoospermic patients (TESA-NOA group, n = 58), [2] TESA in obstructive-azoospermic patients (TESA-OA group, n = 48), [3] and percutaneous epididymal sperm aspiration in obstructive-azoospermic patients (PESA-OA, n = 98). For each experimental group, cycles where AOA was applied (subgroup: activation) were compared with cycles in which AOA was not applied (Subgroup: control). The fertilization, high-quality embryo, implantation, and pregnancy rates were compared among the subgroups.Result(s): For patients undergoing TESA, AOA did not improve ICSI outcomes for either type of azoospermia. However, for cases in which the injected sperm were retrieved from the epididymis, a statistically significantly increased rate of high-quality embryos was observed with AOA.Conclusion(s): Artificial oocyte activation may improve ICSI outcomes in azoospermic patients when epididymal, but not testicular spermatozoa, are injected. (Fertil Steril (R) 2009;92:131-6. (C)2009 by American Society for Reproductive Medicine.)

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 x g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 mu m in diameter, with a dark ooplasm surrounded by three-or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70 degrees C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO(2) at 38 degrees C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.

Structure of the Lining Epithelium of the Cauda Epididymis of the Golden Hamster

The ductus epididymis has roles in the maturation and storage of spermatozoa. The main function of the cauda epididymis is the storage of spermatozoa; however, this region exerts other morphophysiological roles. So, this study was aimed at investigating structural features of the cauda epididymis epithelium, which could indicate roles other than the storage. The relative percentages of the cell types in the epithelium were 74.9, 6.9, 12.5 and 5.6% of principal, clear, basal and halo cells respectively. Large intercellular spaces were seen among the lateral plasmatic membranes of adjacent principal cells or among these cells and others cell types. These spaces were found to be filled with multivesicular bodies, myelin figures, scrolls and debris of membranes or flocculent dense material. Clear cells had the cytoplasms filled with lysosomes (3/4 of basal cytoplasm), and vacuoles and vesicles (1/4 of apical cytoplasm). The observations allowed us to infer that clear cells could act in the process of endocytosis and also in water transfer from the lumen to the interstitium through the epithelium compartment. Moreover, transcytosis may occur at the cauda epididymis of Golden hamster.

Ultrastructure of the epithelium lining of cauda epididymidis in mongrel dogs

The epithelium lining of cauda epididymidis in mongrel dogs was examined by transmission electron microscopy. The epididymal epithelium is pseudostratified with stereocilia and is composed predominantly of principal and clear cells. Therefore, exist basal and apical cells. The principal and clear cells show features suggesting that they may be preferentially involved in absorptive and secretive functions. These results are compared with previously published data on the cauda epididymidis of other mammalian species, in order to understand the significance of the epididymis in sperm maturation.

Effects of altered epididymal sperm transit time on sperm quality

The epididymal sperm transit time seems to have an important role in the process of sperm maturation, and it seems that alterations to the transit can harm the process. The aim of the present work was to evaluate the influence of altered sperm transit time through the epididymis on sperm parameters and fertility of rats, as well as the role of testosterone in the alterations. Sprague-Dawley adult male rats were randomly assigned to four different groups and were treated for 12 days: (i) 10 mu g/rat/day DES, to accelerate the transit; (ii) 6.25 mg/kg/day guanethidine sulphate, to delay the transit; (iii) same treatment as group 1, plus androgen supplementation; (iv) control animals received the vehicles. Guanethidine treatment delayed the sperm transit time through the epididymal cauda, provoking increased sperm reserves in this region. Animals exposed to DES showed an acceleration of sperm transit time in the epididymis, and consequently decreased sperm density in both epididymal regions, the caput-corpus and cauda, and diminished sperm motility. In both cases sperm production was not altered. Testosterone supplementation was able to restore the transit time to values close to normality, as they were higher than in the control rats. The same occurred in relation to sperm motility. Rats exposed to DES presented lower fertility after in utero artificial insemination using sperm collected from the proximal cauda epididymis. Therefore, it was concluded that the acceleration of rat sperm transit time appeared to harm normal sperm maturation, thus decreasing sperm quality and fertility capacity, in an androgen-dependent way.

Effects of guanethidine-induced sympathectomy on the spermatogenic and steroidogenic testicular functions of prepubertal to mature rats

Selective chemical sympathectomy of the internal genital organs of prepubertal to mature male Wistar rats was performed by chronic treatment with low doses of guanethidine. Sympathetic denervation caused an increase in intratesticular progesterone levels in prepubertal and early pubertal rats in addition to a decrease in androstenedione and testosterone levels in prepubertal animals, thus indicating a decrease in the conversion of progesterone into androgen, probably by blocking the steroidogenic enzymatic pathway at the 17 alpha-hydroxylase/17,20 desmolase level. A lower degree of testicular maturation, probably related to reduced androgen activity, was observed in prepubertal and early pubertal sympathectomized rats. Concentration of spermatozoa, on the other hand, was increased in the enlarged cauda epididymidis of late pubertal and mature denervated animals. This result is discussed in terms of the impairment of epididymal mechanisms of seminal emission, fluid resorption and spermatozoal disposal.

Alterações reprodutivas em ratos machos expostos perinatalmente aos frutos da Solanum lycocarpum

This work employed pregnant rats treated with Solanum lycocarpum unripe fruits (10% in diet) from gestation day (GD) 06 to post-natal day (PND) 07, for the evaluation of the sperm number, daily sperm production and epididymal sperm transit time of the male offspring at PND 60 and PND 90. No differences were observed in the daily sperm production (DSP) and sperm number in the testis of the exposed males at PND 60 and PND 90. Also, no alterations were observed in sperm transit time in the caput epididymis of the exposed males at PND 60 and PND 90. However, a reduced sperm transit time was observed in the corpus/cauda epididymis of the experimental males at PND 90. The last data may explain the reduced sperm number observed in the corpus/cauda epididymis of the experimental male rats at PND 90. These data show that the male rats exposed to S. lycocarpum fruits during gestation did not present alterations in testis sperm production and number, however the sperm transit time through epididymis was impaired, resulting in a decreased number of spermatozoa in epididymis cauda. We conclude that S. lycocarpum may cause imbalance on hypothalamus-pituitary gland axis.

Annual reproductive cycle of males of the flat-faced fruit-eating bat, Artibeus planirostris (Chiroptera: Phyllostomidae)

Artibeus planirostris is an endemic species of Phyllostomid bat from the Neotropical region. Some studies have indicated that it exhibits seasonal bimodal polyestry; however, others postulate that it may be able to produce young at any time during the year. Thus, the aim of this study was to evaluate the annual variations in testicular and epididymal parameters of this species in southeast Brazil and try to understand how the reproduction of this species is regulated in this environment. Sixty mature male specimens, collected between June 2009 and May 2010, were submitted to morphometric and immunohistochemical analysis. Our study showed that A. planirostris presented a continuously active pattern of spermatogenesis throughout the year, presenting spermatozoa inside its cauda epididymis in all months, but with two pronounced peaks of spermatogenic production, one in September and other in February. We propose that the males developed these two peaks in order to produce sufficient sperm for the reproduction in a harem system and to synchronize with the female reproductive cycle, which had a bimodal polyestric pattern. Control of this variation is directly linked to the expression of the androgen receptor (AR) in Sertoli cells and to serum testosterone levels, which appear to synchronize to establish these two peaks. In the months preceding the two peaks, the testis have a higher expression of the AR, which possibly stimulates the increase in PCNA, and drives a gradual increase in the testicular parameters. Taken together the results suggest that if sperm storage happens in this species, it is of short duration. © 2013 Elsevier Inc.