Evaluation of males spermiogram in domestic pigs

A microscopic assessment was used to estimate sperm morphological structure in 405 ejaculates from 70 boars according to Blom classification. The classification of spermiogram quality according to a new 6-degree scale was also established. We found, that male spermiograms in domestic pigs were characterized by a large variability both between and within breeds. The estimation of semen quality, only with regard to the mean frequency of occurrence of particular morphological forms of spermatozoa, was not effective for evaluation of individual boar or group of boars. However, the method was effective to evaluate individual ejaculates. The quality classification of the spermiogram enabled to evaluate usefulness of ejaculates graded as good and average for insemination.     

Determining sample size for the morphological assessment of sperm

Morphologic assessment of spermatozoa is an integral component in the analysis of semen. Whether a technician rapidly screening semen quality at a commercial stud, a veterinarian performing breeding soundness examinations, a clinician at a reference andrology laboratory providing auditing or diagnostic services, or a researcher evaluating morphology as a part of a fertility study, it is important to make an informed decision regarding the number of spermatozoa to include in the morphology assessment. Application of basic statistical principles such as the nature of proportions, level of confidence in an observed value, and the interaction of sample size with precision, can and should be used in the decision process. This paper outlines in detail the application of these statistical principles in relation to the morphologic assessment of spermatozoa. Guidelines on how these principles can be utilized in practical situations are discussed. Additionally, methodologies for comparison of results within and between laboratories (an area easily prone to misinterpretation) are reviewed. It is hoped that through the use of these fundamental statistical principles, this paper will bring clarity and delineation to the science of quantifying the morphology of spermatozoa.     

Early puberty in local Naga boar of India: assessment through epididymal spermiogram and in vivo pregnancy

Male Naga pig of India, a miniature breed is known for its meat quality and early puberty. No scientific efforts were made to verify the farmers' view that this breed reaches puberty at around 2 months of age. A preliminary study was, therefore, conducted with the objectives: (a) to find out the age at puberty based on mature spermiogram and in vivo pregnancy and (b) to record the sperm morphology in different parts of the epididymis. Animals were selected from two different age groups: group I aged 53 days and 2.4 kg and group II of 85 days and 3.0 kg. Semen samples collected from different sections of epididymis were analyzed for sperm motility, live spermatozoa, and morphological abnormalities. Motility increased (P<0.01) and live spermatozoa and total morphological abnormalities decreased (P<0.001) from caput through cauda epididymis in both the groups. Sperm motility, live spermatozoa and morphologically normal spermatozoa in each section of the epididymis were higher (P<0.01) in group II than I. Boars with >60% progressive motility, >70% live spermatozoa, <15% total morphological abnormalities and <10% abnormal acrosomes in cauda epididymal spermatozoa were considered mature spermiogram. As per this definition, pigs of group II had only mature spermiogram. In vivo pregnancy confirmation indicated that Naga boar could impregnate female as early as 90 days of age. In conclusion, Naga boar attained puberty by not later than 3 months with 3.0 kg, which is the lowest body weight at puberty in this species reported so far, as reflected by mature epididymal spermiogram and in vivo pregnancy confirmation.     

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: asthenozoospermia

The aim of aneuploidy evaluation in spermatozoa from patients presenting spermatogenesis defects is to identify a relationship between meiotic errors and quantitative or qualitative alterations of spermatogenesis. During the past ten years, the use of fluorescence in situ hybridization (FISH) has permitted the determination of the frequency of numerical chromosome aberrations in different clinical situations. It has been established that infertile males with reduced sperm count and a normal constitutional karyotype have a significantly high risk of aneuploidy in their spermatozoa particularly regarding sex chromosomes. Concerning sperm motility, the data are more controversial. However, patients of severe asthenozoospermia induced by specific morphological deformities involving sperm flagella have a significantly high risk of producing aneuploid spermatozoa.     

Control of the eupyrene-apyrene sperm dimorphism in Lepidoptera

Lepidoptera males bear concomitantly nucleate (eupyrene) and anucleate (apyrene) spermatozoa. Both kinds of spermatozoa reach the spermatheca of inseminated females but only the eupyrene ones fertilize the eggs. The functions of the apyrene spermatozoa are still uncertain. Eupyrene spermatogenesis is regular and highly sensitive to genetic and experimental manipulations while apyrene spermatogenesis is irregular and withstands these manipulations. Both kinds of spermatozoa derive from the same kind of bipotential spermatocytes. The shift of spermatocyte commitment from eupyrene to apyrene spermatogenesis is induced by a haemolymph factor that becomes active just before or after pupation, depending on species. Accordingly, eupyrene spermatogenesis starts during larval instars and stops after pupation while apyrene spermatogenesis begins just before or after pupation, depending on the species, and persists in the imago. The shift is related to shortening of meiotic prophases and blocking synthesis of a meiotic lysine-rich protein fraction in apyrene cells. From spermatogonia proliferation to early spermatocytes, spermatogenesis is a quasi-independent process. Afterwards, it becomes discontinuous and is punctuated by predetermined stations. Progress to a subsequent station is an 'all or none' phenomenon, regulated by cues linked to fluctuations of the main morphogenetic hormones titers. In absence of a particular cue, the cells stop advancing towards the next station and eventually degenerate.     

Synergetic Effects of K,Ca, Cu and Zn in Human Semen in Relation to Parameters Indicative of Spontaneous Hyperactivation of Spermatozoa

We have observed that sperm quality parameters indicative of spermatozoa hyperactivation such are lower “linearity” and “straightness”, and as showed by this research “elongation”, were more pronounced in patients with normal spermiogram compared to the group of men with reduced sperm motility who were undergoing routine in vitro fertilisation. The research encompassed 97 men diagnosed with normozoospermia (n = 20), asthenozoospermia (n = 54) and oligoasthenozoospermia (n = 23). The findings indicate that sperm quality of patients with normal spermiogram diagnosed according to WHO criteria, may be compromised by showing premature spontaneous hyperactivation which can decrease the chances of natural conception. We assessed synergistic effects of multiple chemical elements in ejaculated semen to find if premature spontaneous hyperactivation of spermatozoa can be a sign of imbalanced semen composition especially of elements K, Ca, Cu and Zn. Human semen samples showing low or high baseline status of chemical elements concentrations were found in samples from all three diagnostic groups. However, correlation of K/Ca and Cu/Zn ratios, taking into account samples from all three groups of men, were negative at statistical significance level p = 0.01. We tested if the negative correlation between K/Ca and Cu/Zn ratio works for greater number of semen samples. We found the negative correlation to be valid for 175 semen samples at statistical significance of p = 0.00002. The ratio of K/Ca and Cu/Zn, i.e. increased concentrations of K and Zn in comparison to concentrations of Ca and Cu, were associated with a decrease of “straightness” in the group of men with normal spermiogram and pronounced spontaneous hyperactivation of spermatozoa, implying that these elements act in synergy and that the balance of elements and not their absolute concentrations plays the major role in premature spermatozoa hyperactivation in ejaculated semen.     

Spermiogenesis and sperm ultrastructure in ten species of Loricariidae (Siluriformes, Teleostei)

The current study describes the ultrastructural characteristics of spermatogenesis, spermiogenesis, and spermatozoa in specimens of siluriform taxa Neoplecostominae, Hypoptopomatinae, Otothyrinae, Loricariinae, and Hypostominae. Our data show that the characteristics of spermatogenesis and spermiogenesis and spermatozoa ultrastructure of Neoplecostominae are more common to Hypoptopomatinae and Otothyrinae than to Loricariinae and Hypostominae. Furthermore, Loricariinae and Hypostominae have more characteristics in common than with any other group of Loricariidae. These data reinforce the phylogenetic hypotheses of relationships among the subfamilies of Loricariidae. Considering the available data in Loricarioidei, Loricariidae presents ultrastructural characteristics of spermatogenesis and spermiogenesis that are also observed in Astroblepidae, its sister group. However, the most of the characteristics of spermatozoa ultrastructure found in Astroblepidae are also observed in Scoloplacidae, the sister group of a clade composed of Astroblepidae and Loricariidae.     

Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA

To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.     

La place actuelle de la biopsie testiculaire dans l’exploration de l’homme azoospermique

The interest for testicular biopsy has been severely fluctuating during the development of andrological investigation and treatment. In the fifties and sixties much energy was spend on reading spermatogenesis very accurately and most protocols from that period focussed on the levels at which a stop in spermatogenesis could be observed. This work led to several scores that proved however to have no practical therapeutic value. When F.S.H. could be more accurately measured by R.I.A., the interest for biopsy faded severely. It was only rekindled by the andrological microsurgeons but when in its turn this therapeutic approach lost much of its interest by the T.E.S.E. method, also this aspect of the biopsy lost its value. At the present time, most clinicians dealing with the use of testicular spermatozoa in Assisted Fertilisation Programs, are only interested in knowing whether or not some mature spermatozoa are still present. It was clarified that even in very severely damaged testes a few tubules can still contain a limited amount of spermatozoa.     

Abnormal spermatogenesis at low temperatures in the Japanese red-bellied newt,Cynops pyrrhogaster: possible biological significance of the cessation of spermatocytogenesis

In newt testis, spermatocytes never appear during winter, because secondary spermatogonia die by apoptosis just before meiosis. In the current study, we examined the effect of low temperatures on spermatogenesis. Incubation of newts at low temperatures (8, 12, 15 degrees C) induced defects in spermatogenesis in a temperature-dependent manner. At 8 degrees C, multinucleated giant cells (MGCs) were observed in spermatocytes and spermatogenesis never proceeded beyond meiosis. Although spermatocytes completed meiotic divisions at 12 degrees C, severe cell death was observed in the spermatids. At 15 degrees C both normal and abnormal spermiogenesis were observed. Under these conditions, impaired meiotic synapsis/recombination and down-regulation of the expression of the DMC1 protein, which play pivotal roles in meiotic pairing in eukaryotes, were also observed. Furthermore, to examine the quality of the sperm produced at low temperature for supporting development, artificial insemination was performed. The eggs inseminated with spermatozoa derived from newts kept at 15 degrees C demonstrated a restricted developmental capacity, even though these spermatozoa had an equal capacity for carrying out fertilization to those kept at 22 degrees C. These results suggest that meiosis at low temperatures cause the production of abnormal spermatozoa. Conservation and the significance of this phenomenon in poikilothermic vertebrates living in the temperate zones are also discussed.     

Sperm of Doradidae (Teleostei: Siluriformes)

Spermatic characteristics were studied in 10 species representing several distinct groups within the catfish family Doradidae. Interestingly, different types of spermatogenesis, spermiogenesis and spermatozoa are correlated with intrafamilial groups previously proposed for Doradidae. Semi-cystic spermatogenesis, modified Type III spermiogenesis, and biflagellate sperm appear to be unique within Doradidae to the subfamily Astrodoradinae. Other doradid species have sperm with a single flagellum, cystic spermatogenesis, and spermiogenesis of Type I (Pterodoras granulosus, Rhinodoras dorbignyi), Type I modified (Oxydoras kneri), or Type III (Trachydoras paraguayensis). Doradids have an external mode of fertilization, and share a few spermatic characteristics, such as cystic spermatogenesis, Type I spermiogenesis and uniflagellate sperm, with its sister group Auchenipteridae, a family exhibiting sperm modifications associated with insemination and internal fertilization. Semi-cystic spermatogenesis and biflagellate spermatozoa are also found in Aspredinidae, and corroborate recent proposals that Aspredinidae and Doradoidea (Doradidae + Auchenipteridae) are sister groups and that Astrodoradinae occupies a basal position within Doradidae. The co-occurrence in various catfish families of semi-cystic spermatogenesis and either biflagellate spermatozoa (Aspredinidae, Cetopsidae, Doradidae, Malapturidae, Nematogenyidae) or uniflagellate sperm with two axonemes (Ariidae) reinforces the suggestion that such characteristics are correlated. Semi-cystic spermatogenesis and biflagellate sperm may represent ancestral conditions for Loricarioidei and Siluroidei of Siluriformes as they occur in putatively basal members of each suborder, Nematogenyidae and Cetopsidae, respectively. However, if semi-cystic spermatogenesis and biflagellate sperm are ancestral for Siluriformes, cystic spermatogenesis and uniflagellate sperm have arisen independently in multiple lineages including Diplomystidae, sister group to Siluroidei.     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm–egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.     

An ultrastructural study of spermatogenesis and sperm morula breakdown inArenicola marina (L.) (Annelida: Polychaeta)

Spermatogenesis in the lugwormArenicola marina, in common with other members of Arenicolidae, occurs in the coelomic fluid and results in the formation of discs of mature spermatozoa known as a morulae. Within a morula, individual spermatozoa are connected by a common mass of cytoplasm called the cytophore and therefore make up a syncitium. Immediately prior to spawning, and in response to an endocrine substance known as “Sperm Maturation Factor” (SMF), the structure of the sperm morulae breaks down and free spermatozoa are liberated. These are subsequently spawned from the body cavity. The investigation described here uses transmission electron microscopy to investigate the ultrastructural changes, which accompany spermatogenesis and the breakdown of sperm morulae in response to SMF in vitro. The study demonstrates that the cytophore appears to have a key role both during spermatogenesis and during sperm morula breakdown. The ultrastructure of sperm morulae and of mature spermatozoa is described. The structure of spermatozoa is shown to be primitive with a single flagellum which appears to be coiled at its distal end. The phagocytosis of free spermatozoa by coelomocytes is also described and it is suggested that these may play a role in the resorption of unspawned gametes in vivo.     

Establishment of spermatogenesis in guinea-pigs at puberty

The establishment of spermatogenesis in the guinea-pig was studied by a microscopic method. The spermatogenesis starts before day 16 and the first spermatozoa appear on day 55. Spermatozoa are seen in the tail of the epididymis on day 60 and in the ductus deferens between day 70 and day 80. Relations exist between increase of testicular testosterone and setting of spermatogenesis in the guinea-pig.     

Double spermatogenesis in Chelicerata

Sperm dimorphism is a rare phenomenon in Chelicerata. Until now, it was known only from three species of the opilionid genus Siro (Sironidae, Cyphophthalmi). Fertilizing (eusperm) and nonfertilizing spermatozoa (parasperm) develop in the same cyst and are thus sister cells. The fine structure of the spermatozoa of two species has been examined and is compared here. In contrast to Siro rubens, S. duricorius spermatozoa lack an acrosomal complex. Both sperm types produce a transitional process, a more or less modified flagellum, which is later retracted. Hence, the spermatozoa are aflagellate. Eusperm and parasperm of all three species form highly ordered sperm balls that are stored in the deferent duct. Reviewing and adding new results about the sperm dimorphism in this arachnid taxon provides the basis for some considerations of another enigmatic morphological character found in Uropygi and Amblypygi, i.e., the tubular accessory genital glands that show holocrine extrusion. These glands are suggested to represent modified, infertile derivatives of the testis anlage. Their secretion is produced in a way reminiscent of a strongly degenerated spermatogenesis. Consequently, these products may be regarded as strongly degenerated germ cells representing a line of germ cell development, which has been separated very early in spermatogenesis from the usual line leading to fertilizing sperm cells. This further, although less evident, case of probable dichotomous germ cell development is discussed with respect to the controversial phylogenetic-systematic relationships between Uropygi (Thelyphonida and Schizomida), Amblypygi, and Araneae.     

Abnormal Sperm Parameters in Humans Are Indicative of an Abortive Apoptotic Mechanism Linked to the Fas-Mediated Pathway

The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during spermatogenesis. The presence of other molecular markers of apoptosis has, however, not been shown. More importantly, differences in these markers have not been investigated in men with normal and abnormal sperm parameters. In this study we examine for the presence of the cell surface protein Fas in ejaculated human spermatozoa. Ejaculated spermatozoa (55 samples) were labeled with anti-human Fas antibody and the number of spermatozoa displaying Fas were counted using a fluorescence-activated cell sorter (FACS). In 30/31 (96.8%) normal males (>20 million sperm per milliliter), less than 10% of the spermatozoa were Fas positive. In contrast, 14/24 (58.3%) oligozoospermic samples (<20 million sperm per milliliter) contained more than 10% Fas-positive spermatozoa. Similar differences were observed in men whose spermatozoa had poor motility and morphology. These results indicate that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are clear differences in molecular markers of apoptosis between males with normal and abnormal sperm parameters. We propose that the presence of Fas-labeled spermatozoa in the ejaculate of these men is indicative of an "abortive apoptosis" having taken place, whereby the normal apoptotic mechanisms have misfunctioned, have been overridden, or have not been completed.     

Structure of the testis and spermatogenesis of the viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico

We describe the histological characteristics of the testis and spermatogenesis of the cave molly Poecilia mexicana, a viviparous teleost inhabiting a sulfur spring cave, Cueva del Azufre, in Tabasco, Southern Mexico. P. mexicana has elongate spermatogonial restricted testes with spermatogonia arranged in the testicular periphery. Germ cell development occurs within spermatocysts. As spermatogenesis proceeds, the spermatocysts move longitudinally from the periphery of the testis to the efferent duct system, where mature spermatozoa are released. The efferent duct system consists of short efferent duct branches connected to a main efferent duct, opened into the genital pore. Spermatogenesis consisted of the following stages: spermatogonia (A and B), spermatocytes (primary and secondary), spermatids, and spermatozoa. The spermatozoa are situated within spermatocysts, with their heads oriented toward the periphery and flagella toward the center. Once in the efferent duct system, mature spermatozoa are packaged as unencapsulated sperm bundles, that is, spermatozeugmata. We suggest that the histological characteristics of the testis and spermatogenesis of P. mexicana from the Cueva del Azufre, and the viviparous condition where the spermatozoa enter in the female without been in the water, have allowed them to invade sulfurous and/or subterranean environments in Southern Mexico, without requiring complex morphofunctional changes in the testis or the spermatogenetic process.