Testis Growth Research Articles

A comparison of ultrasound-based testis volume with Prader orchidometry and stability of testis size relative to peers from birth to 28weeks.

Testis volume, an indicator of the reproductive development during minipuberty, is commonly measured by Prader orchidometer, despite ultrasound being the gold standard. Data are lacking on the longitudinal relationship between these two measures and on the stability of boys' relative testis size across infancy. To examine the relationship between ultrasound-based and orchidometer-based testis volume measurements and to assess the stability of relative testis size among individual boys in the study. The Infant Feeding and Early Development study is a longitudinal cohort of healthy infants recruited from hospitals in the Philadelphia area during 2010-2013. We measured testis size from birth to 28weeks in 147 infants using Prader orchidometry (nine study visits) and ultrasound (five study visits). We modeled testis growth, extracted predicted volumes for each boy on each day of the study, and ranked these volumes from smallest to largest. The average testis volume trajectory exhibited linear growth over the first 16weeks followed by slower growth and then a plateau. Prader orchidometry overestimated testis size by almost 3-fold, compared to ultrasound. A range of ultrasound volumes corresponded to each bead size (e.g., bead size of 1 cm3 corresponded to an ultrasound-based volume between 0.11 and 0.87 cm3). Infants changed rankings of median of 22 positions (of 147) across the entire 6-month follow-up. Infants' ranks near birth were highly correlated with their ranks at the end of the study. Consistent with other studies, we found wide variability in testis size during infancy and that Prader orchidometry overestimates testis size. When compared to ultrasound, orchidometry only crudely estimates testis size in this age group. Ultrasound-based volumes generally showed stability in relative testis size across infancy. Accurate measurement of testis size is difficult using orchidometry in infants. This highlights the need for ultrasound for accurate measurement, with a one-time measurement likely sufficient to determine relative testis size across the first 6 months of infancy.

Undescended Testes Growth Potential in Relation to Testis Position from Diagnosis until Puberty.

Background: Testicular volume (TV) and testicular atrophy index (TAI) were used to determine criteria for normal, hypotrophic and atrophic undescended testes (UDT). Objectives: This study aimed to determine changes in TV and TAI in patients with different types of UDT. Materials and Methods: 182 boys (aged 0.3-14.0 years) with 212 UDTs were assessed twice 24 months apart. Testes were unilateral (UCT) or bilateral canalicular (BCT) and intra-abdominal (IAT). Results: At the beginning of the observation, the highest TAI was observed in IAT and the lowest in the BCT group (38.1 vs. 12.5%, p < 0.05). After 2 years, the highest TAI was observed in the BCT and IAT groups (20.5 and 19.1%), while the lowest was in the UCT group (12.0%, p < 0.05). At the beginning and after 2 years, the highest TAI was observed in boys aged < 6 years (25.0%, 18.2%) and the lowest in pubertal boys aged 12-14 years (5.9%, 7.3%, p < 0.05). A total of 78.3% of patients at the beginning and 86.8% at the end of the observation had TAI < 30%. Furthermore, 7% of boys at the beginning and 3% at the end of the observation had TAI > 50%. IATs have the highest testicular growth potential (TGP), while BCTs have the lowest (120.0 vs. 28.6%, p < 0.05). The highest TGP was in boys aged < 3 years (100%, p < 0.05) and boys aged 12-14 years (98.1%, p < 0.05), while the lowest was in boys aged 9-10.9 years (19.5%, p < 0.05). Conclusions: We revealed the continuous growth of UDTs until puberty independently of their position. IATs revealed high growth potential.

Effects of different hormonal treatments on spermatogenesis advancement in hatchery-produced greater amberjack Seriola dumerili (Risso 1810)

In earlier studies, wild-caught greater amberjack Seriola dumerili (Risso, 1810) males reared in sea cages showed gametogenesis impairment and low sperm production and quality. Here, we (a) examined if F1 hatchery-produced males reared in sea cages also exhibit reproductive dysfunctions and (b) evaluated the effects of gonadotropin releasing hormone agonist (GnRHa) administration through injections (GnRHainj) or sustained-release implants (GnRHaimpl), and human chorionic gonadotropin (hGC) injections on spermatogenesis/spermiation enhancement. Fish were given a hormone treatment just prior to the spawning season, and were transferred to land-based tanks, according to an established spawning induction protocol. Blood samples (n = 6) were obtained on Days 0, 7 and 13 after treatment. Testis samples were obtained on Days 0 (n = 4) and 13 (n = 2 per treatment). The fish prior to their transfer from the sea cages to the land-based tanks, exhibited a low gonadosomatic index, altered sex steroid hormone profile and high density of testicular apoptotic cells. After transfer to tanks, there was a general depression of sex steroid plasma levels parallel to an increase in cortisol concentrations. Despite the negative effect on steroidogenesis by the transfer from the sea, the hormonal treatments increased the number of fish from where sperm could be obtained, as well as testis growth, and reduced testicular apoptosis. Treatment with hCG resulted in the most significant changes in spermatogenesis, while GnRHaimpl appeared to induce less intense, but likely longer-lasting effects. The study indicated that F1 hatchery-produced males also exhibited reproductive dysfunctions as wild-caught captive-reared greater amberjack, and that the observed positive effects of the hormone treatments on spermiation/spermatogenesis were likely mediated by factors other than sex steroid hormones.

Testis exposure to unopposed/elevated activin A in utero affects somatic and germ cells and alters steroid levels mimicking phthalate exposure.

Correct fetal testis development underpins adult male fertility, and TGFβ superfamily ligands control key aspects of this process. Transcripts encoding one such ligand, activin A, are upregulated in testes after sex determination and remain high until after birth. Testis development requires activin signalling; mice lacking activin A (Inhba KO) display altered somatic and germ cell proliferation, disrupted cord elongation and altered steroid synthesis. In human pregnancies with pre-eclampsia, the foetus is inappropriately exposed to elevated activin A. To learn how this affects testis development, we examined mice lacking the potent activin inhibitor, inhibin, (Inha KO) at E13.5, E15.5 and PND0. At E13.5, testes appeared similar in WT and KO littermates, however E15.5 Inha KO testes displayed two germline phenotypes: (1) multinucleated germ cells within cords, and (2) germ cells outside of cords, both of which are documented following in utero exposure to endocrine disrupting phthalates in rodents. Quantitation of Sertoli and germ cells in Inha KO (modelling elevated activin A) and Inhba KO (low activin A) testes using immunofluorescence demonstrated activin A bioactivity determines the Sertoli/germ cell ratio. The 50% reduction in gonocytes in Inha KO testes at birth indicates unopposed activin A has a profound impact on embryonic germ cells. Whole testis RNAseq on Inha KO mice revealed most transcripts affected at E13.5 were present in Leydig cells and associated with steroid biosynthesis/metabolism. In agreement, androstenedione (A4), testosterone (T), and the A4:T ratio were reduced in Inha KO testes at E17.5, confirming unopposed activin A disrupts testicular steroid production. E15.5 testes cultured with either activin A and/or mono-2-ethylhexyl phthalate (MEHP) generated common histological and transcriptional outcomes affecting germline and Leydig cells, recapitulating the phenotype observed in Inha KO testes. Cultures with activin A and MEHP together provided evidence of common targets. Lastly, this study extends previous work focussed on the Inhba KO model to produce a signature of activin A bioactivity in the fetal testis. These outcomes show the potential for elevated activin A signalling to replicate some aspects of fetal phthalate exposure prior to the masculinization programming window, influencing fetal testis growth and increasing the risk of testicular dysgenesis.

Testicular catch-up growth in the non-operative management of the adolescent varicocele

Adolescent varicocele is a common urologic condition with a spectrum of outcomes, leading to variations in management. Testicular hypotrophy is a common indication for surgery Routine monitoring may be an appropriate form of management for many adolescents with testicular hypotrophy, as studies have shown that a large proportion of these patients may experience catch-up growth of the ipsilateral testis. Furthermore, there are few longitudinal studies which have correlated patient specific factors to catch-up growth. We aimed to determine the frequency of testicular catch up-growth in adolescents with varicocele while also examining if patient specific factors such as BMI, BMI percentile, or height correlated with testicular catch-up growth. A retrospective chart review found adolescent patients who presented to our institution with varicocele from 1997 to 2019. Patients between the ages of 9 and 20 years with left-sided varicocele, a clinically significant testicular size discrepancy, and at least two scrotal ultrasounds at least one year apart were included in analysis. Testicular size discrepancy of greater than 15% on scrotal ultrasound was considered clinically significant. Testicular size was estimated in volume (mL) via the Lambert formula. Statistical relationships between testicular volume differential and height, body mass index (BMI), and age were described with Spearman correlation coefficients (ρ). 40 patients had a testicular volume differential of greater than 15% at some point during their clinical course and were managed non-operatively with observation and serial testicular ultrasounds. On follow-up ultrasound, 32/40 (80%) had a testicular volume differential of less than 15%, with a mean age of catch up growth at 15 years (SD 1.6, range 11-18 years). There were no significant correlations between baseline testicular volume differential and baseline BMI (ρ=0.00, 95% CI [-0.32, 0.32]), baseline BMI percentile (ρ=0.03, 95% CI [-0.30, 0.34]), or change in height over time (ρ=0.05, 95% CI [-0.36, 0.44]). The majority of adolescents with varicocele and testicular hypotrophy exhibited catch-up growth with observation, suggesting that surveillance is an appropriate form of management in many adolescents. These findings are consistent with previous studies and further indicate the importance of observation for the adolescent varicocele. Further research is warranted to determine patient specific factors that correlate with testicular volume differential and catch up growth in the adolescent varicocele.

Sex Reversal Induced by Dietary Supplementation with 17α-Methyltestosterone during the Critical Period of Sex Differentiation in Oriental River Prawn (Macrobrachium nipponense).

The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis-ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.

European brown hare (Lepus europaeus Pall.) testes weights from different seasons and ages from Bulgaria

Testis weights on 315 European brown hare from different ages were measured during the whole year around in Bulgaria and testicular activity was studied. From the results, we determinate, that the active reproductive season finish at end of August and beginning of September, whereupon follow period of calm when the testis weights were lowest. In September, October, and the beginning of November, the weights of the testes were low. The first indications for starting of the new reproductive season were shown in November, gradually with growth of the testis weights. In January the testis weights reached high levels, as the highest weights were measured in May-June and with a bit lower values in July and August. After this follow low weights, when the calm period started in September and the testis growth is reactive in December. All adult males (above 1 year old, determined by dry eye lens weights, n = 160), had high significant values (p&lt; 0.0001) of testis weights during the whole year, compare with all young ones (under 1 year old, determined by dry eye lens weights, n = 155). The results showed that normal reproductive cycle with periods of activity and calm in male European brown hares from Bulgaria. The distribution of testes weights, compared to the various ages, follows the same sequence, as at the sperm density (we assigned sperm quantity for each individual in the epidydimides to five classes: 1 – no sperm, 2 – very little, 3 – moderately present, 4 – high amount, 5 – massive amount). View of the fact that the density of sperm is defined by the tail of the epididymis, and the weights were measured from the testes, the connection between them confirms the proofs about the existence of a normal reproductive cycle with stages of activity and standstill. The heaviest testes had the biggest quantity of sperm in the epididymis, independently from the season. We defined, that the quantity of sperm and the weights of the testes were in direct dependency (p &lt; 0.0001), as exactly following the line of activity and standstill in the seasons.

Constant High Temperature Promotes Early Changes in Testis Development Associated with Sexual Maturation in Male Atlantic Salmon (Salmo salar L.) Post-Smolts

Sexual maturation of post-smolts is a concern for Atlantic salmon producers, and its occurrence is dependent upon factors such as water temperature and energy availability, among others. The present study was designed to investigate the effect of different temperatures and feeding regimes on testis development and local regulation of spermatogenesis in salmon post-smolts. A 3 × 2 factorial design was used, with three temperatures (8 °C, 12.5 °C, and 18 °C) and two feed regimes (100% and 67% ration). Salmon (1800 parr, initial mean weight 23.1 ± 7.2 g) were reared from 28 October 2018 to 30 May 2019 in a freshwater flow-through system under continuous light (LD24:0), except for a 5-week winter signal (LD12:12) introduced on 4 February 2019. Testis histology, transcription of follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr), and transcription of testis proteins involved in spermatogenesis regulation such as gonadal-soma-derived factors 1 (gsdf1) and 2 (gsdf2), anti-Müllerian hormone (amh), and insulin-like growth factor-3 (igf3), were analyzed. Results showed that high temperature alone (18 °C), irrespective of the feed regime, promoted early presence of type B spermatogonia and reduced transcript levels of the proliferation-inhibitory factor amh in males still considered immature, of groups 18–100% and 18–67% that later matured in high proportion (~100%). This effect was also present to some degree in the group 12.5–100% (40% maturation), and absent in 12.5–67%, 8–100%, and 8–67% (groups with little or no maturation). Later, at onset of rapid testis growth, high temperature was linked to a pronounced downregulation of amh and of the self-renewal factors gsdf1 and gsdf2, as well as to a pronounced upregulation of the proliferation-stimulating factor igf3. Overall, the present findings demonstrate that rearing salmon at high temperatures can stimulate an early activation of the brain–pituitary–gonad axis before actual onset of rapid testis growth, enhancing and accelerating the mechanisms that control initiation and progression of maturation, while the feed regime has a minor impact. This poses a challenge for current salmon aquaculture practices that use sustained high temperature to maximize growth, since these practices increase the risk of early post-smolt maturation.

The adiponectin receptor agonist, AdipoRon, promotes reproductive hormone secretion and gonadal development via the hypothalamic-pituitary-gonadal axis in chickens

Adiponectin is a key hormone secreted by fat tissues that has multiple biological functions, including regulating the energy balance and reproductive system by binding to its receptors AdipoR1 and AdipoR2. This study investigated the correlation between the levels of adiponectin and reproductive hormones in the hypothalamic-pituitary-ovarian (HPO) axis of laying hens at 4 different developmental stages (15, 20, 30, and 68 wk) and explored the effects of AdipoRon (an activator of adiponectin receptors) on the hypothalamic-pituitary-gonadal (HPG) axis and follicle and testicular Leydig cells in vitro and in vivo. The results demonstrated that the adiponectin level was significantly correlated with that of reproductive hormones in the HPO axis (e.g., GnRH, FSH, LH, and E2) in laying hens at 4 different ages. Moreover, AdipoRon could promote the expression of AdipoR1 and AdipoR2 and the secretion of reproductive hormones in the HPG axis, including GnRH, FSH, LH, P4, and T. AdipoRon could also upregulate the expression of genes related to follicular steroidogenesis (STAR, CYP19A1, CYP17A1, and CYP11A1), hepatic lipid synthesis (OVR, MTP), follicular lipid uptake (PPAR-g), and follicular angiogenesis (VEGFA1, VEGFA2, VEGFR1, ANGPT1, ANGPT2, TEK) in the oviposition period, and all of these findings were consistent with the results obtained from in vitro experiments after the transfection of small white follicles (SWFs) with AdipoRon. Furthermore, the results suggest that AdipoRon increases the diameter of testicular seminiferous tubules, the number of spermatogenic cells and sperm production in vivo and enhances the expression of AdipoR1, AdipoR2 and steroid hormones in vitro. Collectively, the findings suggest that AdipoRon could facilitate the expression and secretion of reproductive hormones in the HPG axis by activating its receptors and then improve the growth and development of follicles and testes in chickens.

Evolution of testicular asymmetry during puberty in adolescents without and with a left varicocele.

To assess the evolution of the Testicular Atrophy Index (TAI) in adolescent boys with and without a left varicocele with special attention for the currently postulated cut-off value of 20%. During 2015-2019, 364 adolescent boys aged 11-16 years were recruited. Genital examination and scrotal ultrasonography were repeatedly performed (≥4 month intervals). Testicular volume (TV) was calculated using the Lambert formula (length×width×height× 0.71). TAI was calculated using the formula: [(TVright-TVleft)/TVlargest (right, left)]× 100. The final study population comprised 239 participants, 161 (67.36%) controls and 78 (32.64%) adolescent boys with left varicocele. The mean (sd) number of measurements per participant was 3.82(1.08). A TAI of ≥20% at first measurement occurred in 9.94% and 35.90%, respectively. Of these, only 31.25% and 46.43% had a TAI of ≥20% at the last measurement, respectively. Nevertheless, the risk of ending up with a TAI of ≥20% was significantly higher if a TAI of ≥20% was recorded at first measurement (P= 0.041 and P= 0.002, respectively). The normalisation rate did not differ significantly between the groups (P= 0.182). Normalisation occurred most frequently in Tanner Stages III and IV. Normalisation was mostly (≥74%) due to catch-up growth of the left testis, in contrast to growth retardation of the right testis, in both groups. The TAI seems to be a fluctuating parameter. A TAI of ≥20% is a phenomenon seen in boys with and without varicocele but is more common in boys with varicocele. Although normalisation of a high TAI is frequently seen, both adolescent boys with and without a left varicocele who have an initial TAI of ≥20% have a higher risk of a TAI of ≥20% in the future. As the TAI is a fluctuating parameter during pubertal development, it's use as indicator for varicocelectomy based on a single measurement during pubertal development is questioned.

Polygenic co-expression changes the testis growth, hormone secretion and spermatogenesis to prompt puberty in Hu sheep

In most male mammals, testis undergoes increased proliferation activity and the onset of spermatogenesis during pubertal development. However, their gene expression patterns and roles in sheep remain unclear. Therefore, we used Illumina Hiseq 2000 sequencing to identify differentially expressed genes (DEGs) from 0, 30, 60, 90, 120, 150 and 180 postnatal days and characterize the transcriptional level of sheep testicular development. Among them, the DEGs changed the most in 90–150 stages. a total of 2546 (1454 up and 1092 down) and 6867 (4683 up and 2184 down) DEGs were identified in D120 vs. D90 and D150 vs. D120, respectively. Functional enrichment analysis revealed that in earlier pubertal development, testis showed higher gene expression in organ morphogenesis, vasculogenesis, neurogenesis and hormone secretion, while in later pubertal development, genes with higher expression mainly concentrated in regulating spermatogenesis process. These results indicated that testis development undergoes the transition from organ growth to spermatogenesis and the genes related to hormone secretion were expressed highly earlier than spermatogenesis during pubertal process. In addition, we found several genes such as ZFP36, TNF, HSD3B1, HSD11B2 played key roles in androgen secretion and SPAG family, SYCP family, SPATA family, SPO11, CABYR, TNP1, TNP2 and CFAP43 performed functions during spermatogenesis process. Taken together, multi-genes cooperation prompt testis development in pubertal process. Our transcriptional atlas of sheep testis provides a comprehensive insight about testicular development and pubertal process.

Morpho-Histology and Morphometry of Chicken Testes and Seminiferous Tubules among Yellow-Feathered Broilers of Different Ages.

Simple SummaryTestes are important male reproductive organs that in chickens have been greatly investigated, from pre-hatch to after sexual maturity. The present study investigated the changes in components that occur during growth, and evaluated morphometry of the seminiferous tubules (ST), as well as gonadosomatic index (GSI) in Gallus domesticus at different age stages. The left and right testes were harvested from 70 chickens, then fixed in alcoholic acetate formalin (AAF) fixative solution, and hematoxylin- and eosin-stained tissues were used for microscopic observations. The results revealed that the left testis (LT) and the right testis (RT) exhibited fuzzy ST features, with apoptotic resorption of many tubules observed in both testes of 1-wk-old chicks only. ST formation was completed at 1 month, with an increase of all morphometric parameters in both testes until sexual maturity (3-mo-old): the age at which we recorded the greatest GSI. This study provides details on ST apoptotic resorption, which is a process not yet reported in existing publications, as well as ST morphometry and GSI, from a juvenile stage of growth towards sexual maturity. This can serve as reference material and also as a data update to better understand the morpho-histological changes that occur in chicken testes during growth.Unlike in many mammals, poultry testes are found in the abdominal cavity where they develop and perform spermatogenesis at high body temperature. Scarce reports among current publications detail the growth of testes and ST morphometry among juvenile chicks. Therefore, this study aims to investigate changes in components occurring in Gallus domesticus testes, by assessing the GSI and morphologically and histologically evaluating the testes and ST morphometry from 1-wk- to 4-mo-old. Right and left testes were collected from 70 healthy chickens divided into seven age-related groups (n = 10) and then immersed into the alcoholic acetate formalin (AAF) fixative solution. Hematoxylin- and eosin-stained tissues were used for microscopic observations. The findings revealed that both testes exhibited smooth features from 1-wk-old to 1-mo-old, and thereafter showed a consistent increase in vascularization until 4-mo-old. Histologically, both testes exhibited unclear ST, with ST apoptotic resorption observed in the 1-wk-old chicks. Until 1-mo-old, ST formed and few spermatogonia differentiated into primary spermatocytes, with all spermatogenic cells observed at 3-mo-old, i.e., sexual maturity. These findings suggest that both testes develop in analogy, and their sizes including increases in length and diameter are related to the spermatogenic activity in the ST. Subsequently, ST resorption by apoptosis is assumed to participate in the physiological mechanism regulating germ cells (GC). Finally, the GSI tended to increase with growth.

Allometric growth of non-carcass components and hind limb tissue composition in Saanen goat kids reared under natural and artificial systems

The aim was to investigate the growth of certain non-carcass components and hind limb tissues using allometric coefficients in Saanen goat kids reared under natural and artificial systems and slaughtered at 80 days or 120 days of age. 42 Saanen kids were used in the study [(24 artificial reared kids (SA-80: 12 kids, SA-120: 12 kids), 18 natural reared kids (SA-80: 9 kids, SA-120: 9 kids)] for determination of indicated parameters. The effect of rearing system was not significant in weights of non-carcass components and hind limb tissue compositions, which indicates that, even though they were fed with milk replacers, artificial reared kids can have similar growth performance when they slaughtered at similar age with natural raised kids. However, slaughter age had significant effect on many non-carcass components, since the weight of the organs increases in parallel with animal’s live weight. It is seen that allometric growth of non-carcass parameters, except skin and testis in both rearing groups and stomach and intestines in natural rearing, was slower (b

The changing landscape of immune cells in the fetal mouse testis.

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80+ cells were the most abundant, comprising 90% of CD45+ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80−/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206−), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00418-022-02129-6.

P-081 Effect of chemotherapy (cytarabine) and acute myeloid leukemia on the development of spermatogenesis, spermatogonial microenvironment and inflammatory factors at the adult age of immature-treated mice

Abstract Study question How do AML and cytarabine affect the development of spermatogenesis, germ cell microenvironment and testicular inflammatory factors at the adult age of immature-treated mice? Summary answer AML and cytarabine significantly damaged the normal histology of the seminiferous tubules and impaired different stages of spermatogenesis, testicular growth/inflammatory factor in the adult age. What is known already The effect of different types of cancers on male fertility was reported. AML constitutes around 20% of the diagnosed childhood leukemia. Cytarabine is used as anti-AML treatment. Cytarabine is involved in reduction of testes growth and induces testicular atrophy in adults. In adult AML patients, it was shown that AML affects semen parameters even before chemotherapy treatment. Furthermore, a significant reduction in semen quality of thawed cryopreserved semen from leukemia patients compared to control patients was reported. However, the effect of AML and cytarabine before puberty on the development of spermatogenesis in adult age was not yet reported. Study design, size, duration Two-week-old males C57/BLACK mice were used. 1. Control group – injected with saline. 2. AML group – injected (intraperitoneal; i.p) with 3x104 AML cells/100 ml (murine C-1498 AML cell line). 3. Cytarabine group - three injections of cytarabine (140 mg/kg/100 ml) were performed every 12 hours. 4. AML+cytarabine group – cytarabine was i.p injected 24 hours after AML injection (injections were performed every 12 hours 3 times). Mice were sacrificed 1,2,3,4,5 weeks after AML injection. Participants/materials, setting, methods Testes were removed, weighted and fixed in Bouin’s solution for histological evaluation, or kept at -70°C for RNA extraction. The presence of premeiotic (SALL4, PLZF), meiotic (CREM-1) and postmeiotic (ACROSIN) cells were examined by immunofluorescence staining analysis and/or RNA expression by qPCR analysis. Main results and the role of chance We have successfully developed a system of AML in immature mice by i.p injection of C1498 cells. Cytarabine prolonged life of AML-treated mice from 2.5 weeks to 4 weeks. AML had no effect on both body and testicular weight. However, cytarabine significantly reduced testicular weight (around 50% of the control in the first 3 weeks after treatment, and around 30% of the control from 3-4.5 weeks after treatment; p &amp;lt; 0.001). One and/or 2 weeks after the treatment showed that both AML and cytarabine alone or in combination significantly decreased seminiferous tubules normal histology compared to control, (30%, 20%, 10% respectively) (p &amp;lt; 0.001). Cytarabine, but not AML significantly increased the expression of PLZF and SALL4 after 2 weeks compared to control (p &amp;lt; 0.001). However, each treatment significantly increased the percentages of seminiferous tubules with apoptotic cells compared to control (p &amp;lt; 0.05). AML or cytarabine significantly decreased the percentages of tubules with meiotic and postmeiotic markers compared to control (p &amp;lt; 0.05). Also, AML or cytarabine significantly decreased the testicular protein levels of GDNF, CSF-1 (p &amp;lt; 0.01) but did not affect LIF compared to control. Furthermore, AML or cytarabine significantly decreased the protein levels of IL-10. However, AML significantly increased IL-6 when cytarabine increased it compared to control (p &amp;lt; 0.01). Limitations, reasons for caution Animal model of leukemia may behave different from human model. Wider implications of the findings We successfully developed AML disease in immature mice, and demonstrated its impairment the normal histology of the seminiferous tubules and spermatogenesis in the adult age. We showed that AML and cytarabine affect testicular microenvironmental and inflammatory factors. Our results may assist in development future therapeutic strategies for male fertility preservation. Trial registration number NON

CARBONATED BEVERAGES AFFECT LEVELS OF ANDROGEN RECEPTOR AND TESTOSTERONE SECRETION IN MICE.

This work aimed to study the influences of carbonated beverages (CBs) on the testis growth and the expression levels of androgen receptor (AR) of mice. Two experimental groups of 30 mice each PEP-1 and PEP-2 drank 50% and 100% Pepsi-Cola, respectively for 15 days. Other 2 experimental groups of 30 mice each COC-1 and COC-2 drank 50% and 100% Coca-Cola, respectively for 15 days. The control group (CG) of 30 mice drank water. Bilateral testes were harvested aseptically on days 0, 5, 7, 10, 13 and 15. Real-time PCR and Western blot were implemented to detect levels of androgen receptor (AR) mRNA and protein in testis tissues. Testes masses of PEP-2, COC-1 and COC-2 were greater than those of PEP-1 and CG (P < 0.05). On day 15, testis longitudinal diameter (TLD) of CBs-treated mice was increased as compared to CG. TLD, testes transverse diameters (TTD) and AR proteins levels of PEP-2 and COC-2 were increased in comparison with CG (P<0.05). Serum testosterone concentrations of PEP-2 were higher than that of COC-1 and CG (P < 0.05). Levels of AR mRNAs of four CBs-treated mice were increased by 60.18%, 67.26%, 65.93% and 78.76%. A high concentration of Coca-Cola and Pepsi-Cola could raise TLD and TDD, enhance testosterone secretion, and increase serum EGF concentrations.

Metabolic cost of development, regeneration, and reproduction in the planarian Schmidtea mediterranea

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O2 would vary by regeneration day for both sexual and asexual S. mediterranea, reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O2 in sexuals was highest in mature controls at ~23 μl O2/g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O2 of ~10–12 μl O2/g/h. The sum of V̇O2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1–6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O2 differences between sexual and asexual strains.

Neurotransmitters trigger water vole testes growth as days get longer

Success is often about timing. Whether departing to migrate or settling down to hibernate, animals are in tune with the seasons to be certain that they don't misjudge when to embark on essential life experiences. And coordinating the arrival of their next litter is equally critical. ‘To ensure the survival of mothers and juveniles, many species restrict sexual activity to a limited time window, which leads to synchronization of births when environmental conditions are most favorable’, says Matthieu Keller from the CNRS – INRAE Université de Tours, France. And many small creatures effectively put their gonads on ice, to conserve energy until spring, when the days begin growing longer. Knowing that two neurotransmitters – kisspeptin and RF-amide-related peptide 3 (RFRP3), produced in the brain – play a key role in triggering the seasonal growth of many animals’ gonads ready for breeding at the right time of year, Keller and his colleagues went in search of male European water voles (Arvicola amphibius) to find out whether their testes and scent glands flourish as the days grown longer and whether the regrowth of their gonads is coordinated by the two neurotransmitters.Although trapping the voles wasn't technically challenging – ‘you just have to dig and place the trap’, says Kévin Poissenot (INRAE – CNRS Université de Tours) – locating the animals’ burrows with Chantal Moussu (INRAE – CNRS Université de Tours) was tricky, and braving the harsh mountain winters where the voles reside, 25 km from Clermond-Ferrand, France, took some courage. Together, over the 17 months from February 2019 to May 2020, the pair visited the voles, collecting males to find out how the size of their testicles varied.Over the seasons, the water voles’ testes increased in size from 0.06% of body mass (February) to almost 0.4% body mass in June – that's a 6-fold increase – before withering again during the late summer and autumn. In addition, the voles’ scent glands developed in the late winter and spring before shrivelling in late summer and autumn. The team also checked the pituitary, the region of the brain that produces hormones that might coordinate preparations for the breeding season, and found that it too grew, reaching its largest in April before dwindling to its smallest in November. In addition, Poissenot analysed the water voles’ hypothalamuses, which produce many key hormones, and found that the production of kisspeptin – a key signalling molecule that stimulates reproductive function – fluctuated over the year, peaking in February and November, while cells producing the RFRP3 signalling molecule only appeared in the voles’ brains between June and September.But what was driving the males’ dramatic seasonal transformation? After collecting adult males in December 2020, the team transported them back to the lab, providing some animals with 8 h per day of light, to simulate winter, while others were fast-tracked directly to long summer days (16 h per day of light). After 7 weeks, the team checked the testes of the water voles basking in long summer days, and they were almost twice the size (0.44% body mass) of their counterparts that were stuck back in winter (0.26% body mass).It seems that seasonal changes in day length are the key to driving the physical changes undergone by male water voles in preparation for the breeding season, although the team is a little puzzled by the apparent decline in the kisspeptin neurotransmitter at the time when the voles might need more. However, they suggest that kisspeptin could accumulate in the brain over winter – when it isn't released – ready to spring into action, triggering the regrowth of the males’ testes when the sun returns.

Rearing system influences the testicular development, semen quality and spermatogenic cell apoptosis of layer roosters

The objective of this study was to compare the testicular development, semen quality, and spermatogenic cell apoptosis of roosters reared in colony, single, and large cages. Rohman parental layers (n = 540) were randomly allocated into cages of rearing system groups (135 males and 405 females). The experimental period was 70 to 210 d of age. We compared testicular development and plasma main reproductive hormones (Follicle-stimulating hormone; Luteinizing hormone; Testosterone; Estrogen2;) from d 70 to 210 of roosters among the three systems. In addition, routine semen quality indexes, apoptosis of testicular spermatogenic cells and sperm apoptosis of breeding roosters under three rearing systems on d 175 and d 210 were evaluated. Roosters during the growing period (from d 70 to 140) have rapid testis growth and increasing main reproductive hormones in plasma. At the peak of sexual maturity (d 210), in colony cage, the females have a positive effect and promote the testis development of males. However, the stocking density in colony cage has no effect on testicular development; compared with the single and large cage. Roosters reared in the natural mating system had better semen quality, particularly in semen volume, density, and viability; the hatching % of fertilized eggs and healthy chicks were higher for the colony than single and large cages. Furthermore, the sperm density was higher for colony than single and large cages, which was related to the apoptosis of spermatogonia and spermatocyte, not the apoptosis of mature sperm. This study provided the basic data for the reproductive performance research of chicken reared in the colony cages.

RNA-Seq Implies Divergent Regulation Patterns of LincRNA on Spermatogenesis and Testis Growth in Goats.

Long intergenic non-coding RNAs (lincRNAs) regulate testicular development by acting on protein-coding genes. However, little is known about whether lincRNAs and protein-coding genes exhibit the same expression pattern in the same phase of postnatal testicular development in goats. Therefore, this study aimed to demonstrate the expression patterns and roles of lincRNAs during the postnatal development of the goat testis. Herein, the testes of Yiling goats with average ages of 0, 30, 60, 90, 120, 150, and 180 days postnatal (DP) were used for RNA-seq. In total, 20,269 lincRNAs were identified, including 16,931 novel lincRNAs. We identified seven time-specifically diverse lincRNA modules and six mRNA modules by weighted gene co-expression network analysis (WGCNA). Interestingly, the down-regulation of growth-related lincRNAs was nearly one month earlier than the up-regulation of spermatogenesis-related lincRNAs, while the down-regulation of growth-related protein-coding genes and the correspondent up-regulation of spermatogenesis-related protein-coding genes occurred at the same age. Then, potential lincRNA target genes were predicted. Moreover, the co-expression network of lincRNAs demonstrated that ENSCHIT00000000777, ENSCHIT00000002069, and ENSCHIT00000005076 were the key lincRNAs in the process of testis development. Our study discovered the divergent regulation patterns of lincRNA on spermatogenesis and testis growth, providing a fresh insight into age-biased changes in lincRNA expression in the goat testis.