Cell proliferation in the seminiferous and epididymal epithelia of Sus domesticus

It is important to understand the proliferative activity of the different structures of the male reproductive apparatus in livestock species, such as Sus domesticus, to ensure reproductive efficiency. The main aims of this study were (a) to evaluate the proliferative activity of the spermatogonia in the different stages of the seminiferous cycle and (b) to study the cell proliferation in the epididymal epithelium in each region, identifying the different cells involved. For this, the testes and epididymis of three healthy, sexually mature Sus domesticus boars were used. The organs were processed for light microscopy, and immunohistochemical techniques were used to detect proliferating cell nuclear antigen. The cells immunostaining positively and negatively for proliferating cell nuclear antigen were counted and several parameters and indexes were calculated to evaluate the proliferation in both epithelia, taking into account the stage of the seminiferous epithelium cycle, and, in the case of the epididymal epithelium, the different regions and cells are the same. Finally, a contrast analysis of equality between pairs of means was carried out followed by a least significant differences test, in which differences were considered significant at P < 0.05. In the seminiferous epithelium, the greatest total number of spermatogonia and proliferating spermatogonia was observed in the postmeiotic stages (mainly VII and VIII). The proliferation index of the spermatogonia increased from the meiotic to postmeiotic stages. As regards the epididymal epithelium, the total proliferation index was higher in the caput. In each region, the clear and principal cells showed the highest proliferation index with respect to the total number of cells counted, whereas the proliferation index of each cell with respect to the same type was higher in the clear cells, followed by the narrow and principal cells. In conclusion, the proliferative activity of spermatogonia in the seminiferous epithelium of Sus domesticus is stage-dependent, and mainly occurs in the postmeiotic stages. In the epididymal epithelium, proliferative activity takes place in several cell types and is dependent on the anatomical region of the epididymis. We think that these results may be of importance for understanding the pathologic or reproductive processes in which cell proliferation is involved in the male reproductive system.     

Quantitative immunocytochemical studies on the gonadotrophs isolated from the pituitary of the male rat

Summary Quantitative studies on the population of the gonadotropic cells in the pituitary of adult male rats were performed after trypsic dissociation of the pituitary glands and immunoenzymatic staining with anti--LH or anti--FSH antisera. Number, area and extinction of labelled cells were measured by use of an image analyser and a cytophotometer. The gonadotrophs represent approximately 14% of the pituitary cells. The mean area of gonadotrophs is significantly larger after staining with anti--LH serum than after staining with anti--FSH serum. Planimetric measurement of the gonadotrophs reveals their variability in size ranging between 30 and 160 m². Moreover, the size distribution depends on the staining serum used: more numerous small-sized cells (<75 m²) are stained with anti--FSH serum than with anti--LH serum, which conversely stains more numerous large-sized cells. Cytophotometric measurements indicate that immunostaining varies greatly among cells of the same size class and that the staining intensity appears to increase according to the cell size. These results emphasize the morphofunctional heterogeneity of the gonadotropic cell population.     

Toxoplasma gondii: Reproductive parameters in experimentally infected male rats

Toxoplasma gondii is a protozoan parasite that is globally widespread and infects man and animals. With the aim of studying the influence of toxoplasmosis on male reproductive parameters, we investigated sperm motility, concentration and morphology of male rats experimentally infected by T. gondii. The GT F1 strain of T. gondii tissue cysts were fed at a dose of 5 × 10³ tissue cysts per rat by oral gavage in an experimental group of 42 healthy adult male Wistar rats, while 42 male rats were used as controls. On days 10, 20, 30, 40, 50 and 60 post-inoculation (p.i.) 7 rats from each group were anesthetized. The body weight of each animal was recorded, then epididymis and testes were immediately removed, weighed and semen evaluation was undertaken. Weight of the right epididymis was significantly decreased on day 30 p.i., sperm motility was significantly decreased on days 10, 20, 30, 40, 50 and 60 p.i. and sperm concentration was significantly decreased on days 10, 30, 40 and 60 p.i. A marked increase of sperm abnormalities was noticed on days 30 and 40 p.i. No pathological lesions were detected either in the pituitary gland or the testes. In this study it was found that toxoplasmosis can affect main reproductive parameters in male rats, which are the most predictive of their fertilizing capacity.     

Purinergic signalling in the reproductive system in health and disease

There are multiple roles for purinergic signalling in both male and female reproductive organs. ATP, released as a cotransmitter with noradrenaline from sympathetic nerves, contracts smooth muscle via P2X1 receptors in vas deferens, seminal vesicles, prostate and uterus, as well as in blood vessels. Male infertility occurs in P2X1 receptor knockout mice. Both short- and long-term trophic purinergic signalling occurs in reproductive organs. Purinergic signalling is involved in hormone secretion, penile erection, sperm motility and capacitation, and mucous production. Changes in purinoceptor expression occur in pathophysiological conditions, including pre-eclampsia, cancer and pain.     

Ultrastructural immunocytochemical localization of LH and FSH in the pituitary of the untreated male rat

Summary Rapid freeze-substitution fixation was employed in immunocytochemical studies on the localization of LH and FSH in the typical gonadotrophs of the anterior pituitary in the untreated male rat; a modification of a recently described ferritin antibody method (Inoue et al. 1982) was used in these studies. It was shown that rapid freeze-substitution fixation provides good preservation not only of the ultrastructure but also of the antigenicity. Both LH and FSH were clearly demonstrated in the same gonadotrophic cells, but the subcellular localization of these gonadotrophins differed: (i) LH was mainly located in small secretory granules, 250–300 nm in diameter; (ii) FSH was mainly present in large secretory granules, up to 500 nm in diameter. In the pituitary gland of the adult male rat, all gonadotrophs that react to antibodies against gonadotrophins are characterized by small and large secretory granules. Other types of cells of the anterior pituitary containing either small secretory granules or resembling corticotrophs with secretory granules assembled at cell periphery did not react to either anti-LH beta or anti-FSH beta serum.For light microscopy, the peroxidase antibody method was used. All of the gonadotrophin-positive cells contain both LH and FSH. None of the pituitary cells reacted to antibody against only one gonadotrophin. However, some cells are LH-rich while other cells are FSH-rich.     

Adrenocortical cell transplantation in scid mice: the role of the host animals’ adrenal glands

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals’ own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals’ adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals’ adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.     

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus)

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the world's most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx.Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness^®Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness^®Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50×). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness^®Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.     

Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.     

Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells

Summary During the development of the spermatogenous cells, the pteridophyteCeratopteris richardii produces three structurally well-defined microtubule organizing centers (MTOCs). The blepharoplast, a spherical body that occurs during the last two spermatogenous divisions, organizes two microtubule (MT) arrays, one associated with a nuclear indentation and the other that organizes the spindle apparatus for the final divisions. After the last spermatogenous division, the blepharoplast reorganizes to produce two new putative MTOCs: the lamellar strip (LS) of the multilayered structure (MLS), which apparently organizes the spline microtubule array, and an amorphous zone (AM), that connects the basal bodies. Thin and semi-thin sections of this tissue were probed with antisera which recognize MTOCs in lower eukaryotes and animals to determine if any of these structures contain MTOC-associated proteins or epitopes recognized by monoclonal antisera. Gamma tubulin antibodies, which recognizeonly the minus ends of MTs in mammalian cells, label along the MT in all arrays found in the pteridophyte spermatogenous cells. Kinetochore MTs are unlabelled near the kinetochore, however. The monoclonal antibodies MPM-2 and C-9, that recognize centrosomal and nuclear epitopes in mammalian cells, label the interphase nucleus, the cytoplasm of mitotic cells, and the blepharoplast during both nuclear indentation and spindle formation. Double labelling of the blepharoplast-containing cells with anti-tubulin and either MPM-2 or C-9 reveals that the blepharoplast-associated fluorescence is the focus of the tubulin arrays. Centrin labels the reorganizing blepharoplast, the MLS, the AM, and a stellate pattern in the transition region of the flagella. These data indicate the usefulness of the structurally well-recognized MTOCs in pteridophyte spermatogenous cells in investigation of land plant MTOCs.     

The relevance of various tests for the study of specificity in immunocytochemical staining: a review

Factors determining the specificity of immunocytochemical (ICC) tissue stainings as well as the various tests to study these factors are discussed. Since every specificity test only deals with particular aspects of the ICC procedure, a practical sequence of known test methods is proposed, which enables the determination of the specificity of the ICC tissue staining and, after possibly needed antiserum purification steps, may result in a monospecific staining. It is made clear that such a sequence has always to include a tissue-spectrum affinity test, in which the spectrum of tissue antigens is controlled for antibody binding. A variety of such tests, consisting of separation of tissue compounds, fixation, and ICC detection, are discussed as well as their pros and cons with respect to their predictability for the actual serum specificity in the tissue section.     

Light-microscopic immunocytochemical localization of fibronectin in the developing rat lung

Summary The development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.     

Extraglandular synthesis of accessory reproductive gland components in male Melanoplus sanguinipes

The accessory reproductive glands (ARG) of the male migratory grasshopper, Melanoplus sanguinipes, are able to accumulate injected labelled ARG protein from the haemolymph. Accumulation is slight in the ARG of 2-day-old virgins, or 7-day-old allatectomized (CA^?) insects. The ARG of 7-day-old virgins, or 7-day-old CA^? insects treated with synthetic juvenile hormone, accumulate about 1.5 times more label than those of 2-day-old insects in a 24-hr period. The ARG of recently mated males accumulate almost four times more label than those of 2-day-old controls. Immunoprecipitation studies indicate that about one fifth of the labelled protein is accumulated unchanged.The fat body and haemolymph contain proteins which are precipitable by antiserum to whole ARG homogenate. The concentration of these proteins in the fat body increases after removal of the ARG, or after copulation. It is concluded that the fat body synthesizes certain proteins which are accumulated by the ARG. Both the synthesis and the accumulation of these proteins are regulated by the corpora allata.     

Structural alterations in the male reproductive system of the freshwater crayfish, Cherax quadricarinatus (Decapoda, Parastacidae)

No diseases affecting reproductive performance have been previously reported in freshwater crayfishes. This study aims to characterise one reproductive system abnormality found in males of Cherax quadricarinatus reared in captivity. Fifteen adult males of C. quadricarinatus (70-110 g) were purchased from San Mateo S.A. farm (Entre Ríos, Argentina) each season during 2007. Macroscopic analysis showed that 26.6% of the animals sacrificed in winter presented brownish distal vasa deferentia. Histological analysis showed different levels of structural abnormality in the epithelium of the vasa deferentia and spermatophore. Granular and hyaline haemocytes were identified within the vasa deferentia but no significant differences were found in the sperm count between normal and brownish vas deferens. Histological analysis of the crayfishes sacrificed in autumn also showed these modifications in 22% of the animals, however, they did not show the brownish colour under macroscopic analysis. The similarities between the male reproductive system syndrome in shrimps and the abnormalities found in C. quadricarinatus are notable. An unspecific response to thermic stress is a possible explanation of these structural alterations.     

Identification of pheromone-like compounds in male reproductive organs of the oriental locust Locusta migratoria

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.     

Adrenocortical cell transplantation in scid mice: the role of the host animals' adrenal glands

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals’ own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals’ adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals’ adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.     

人工巢箱条件下杂色山雀的巢位选择及其对繁殖成功率的影响

杂色山雀(Parus varius)是一种分布区域极其狭窄的小型森林洞巢鸟类,种群数量稀少,在中国大陆仅见繁殖于辽宁省的东南部山区和毗邻辽宁的吉林省西南部山区,为当地留鸟.为了探讨巢址选择对杂色山雀繁殖的影响,找出影响杂色山雀繁殖成功率的巢址选择方面的主要因子,于2009-2011年3-7月,在辽宁省仙人洞国家级自然保护区通过悬挂人工巢箱,进行杂色山雀对人工巢箱的巢址选择以及不同的巢址对杂色山雀繁殖成功率的影响研究.研究表明:杂色山雀多在针阔混交林中活动和繁殖,对位于赤松与蒙古栎混交林中的巢箱有一定偏好.野外共发现24巢杂色山雀在人工巢箱中繁殖,其中15巢繁殖成功,9巢繁殖失败.对杂色山雀利用的巢址样方主成分分析表明,巢位因子( 19.826％)、乔木因子(17.571％)、灌木因子(13.11％)、光照因子(11.587％)、隐蔽因子(10.562％)和边缘效应因子(7.572％)是影响杂色山雀对人工巢箱选择的重要因子.利用巢箱与对照巢箱相比,两者在距水源距离、距路距离、乔木平均高度、灌木平均高度和植被类型这5个环境变量上存在显著差异.繁殖成功巢与繁殖失败巢相比,繁殖成功的巢箱所在位置距路稍远,坡度较高,乔木最大高度较高,灌木盖度略低于繁殖失败巢箱.人为干扰和天敌捕食是造成杂色山雀繁殖失败的主要原因.以上研究结果表明,巢向偏南、距地面2 m以上、周围乔木高大、灌木平均高度大于1.5m、盖度在45％-55％之间、距水源20 m左右、距路20 m以外的位于针阔混交林边缘的巢箱是杂色山雀繁殖的最优巢址.目前杂色山雀的种群数量还很稀少,希望本研究能对这一珍稀鸟类的保护提供重要参考.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.