CircSry regulates spermatogenesis by enhancing γH2AX expression via sponging miR-138-5p

Sry on the Y chromosome is the master switch in sex determination in mammals. It has been well established that Sry encodes a transcription factor that is transiently expressed in somatic cells of male gonad, inducing a series of events that lead to the formation of testes. In the testis of adult mice, Sry is expressed as a circular RNA (circRNA) transcript, a type of noncoding RNA that forms a covalently linked continuous loop. However, the physiological function of this Sry circRNA (circSry) remains unknown since its discovery in 1993. Here we show that circSry is mainly expressed in the spermatocytes, but not in mature sperms and Sertoli cells. Loss of circSry led to the reduction of sperm number and the defect of germ cell development. The expression of γH2AX was decreased and failure of XY body formation was noted in circSry KO germ cells. Further study demonstrates that circSry regulates H2AX mRNA indirectly in pachytene spermatocytes through sponging miR-138-5p. Our study demonstrates that, in addition to its well-known sex-determination function, Sry also plays important role in spermatogenesis as a circRNA.

Ejaculate Allocation and Sperm Characteristics Differ among Alternative Male Types in a Species of Fish with Cooperation and Competition among Unrelated Males

Sexual selection arising from sperm competition has driven the evolution of immense variation in ejaculate allocation and sperm characteristics not only among species, but also among males within a species. One question that has received little attention is how cooperation among males affects these patterns. Here we ask how male alternative reproductive types differ in testes size, ejaculate production, and sperm morphology in the ocellated wrasse, a marine fish in which unrelated males cooperate and compete during reproduction. Nesting males build nests, court females and provide care. Sneaker males only “sneak” spawn, while satellite males sneak, but also help by chasing away sneakers. We found that satellite males have larger absolute testes than either sneakers or nesting males, despite their cooperative role. Nesting males invested relatively less in testes than either sneakers or satellites. Though sneakers produced smaller ejaculates than either satellite or nesting males, we found no difference among male types in either sperm cell concentration or sperm number, implying sneakers may produce less seminal fluid. Sperm tail length did not differ significantly among male types, but sneaker sperm cells had significantly larger heads than either satellite or nesting male sperm, consistent with past research showing sneakers produce slower sperm. Our results highlight that social interactions among males can influence sperm and ejaculate production.

Female accessory gland fluid promotes sperm survival in yellow dung flies

Female and male reproductive traits co-evolve through pre- and post-copulatory sexual selection and sexual conflict. Although males typically transfer many sperm during copulation, only a small proportion reach the fertilization site because females often actively or passively reduce sperm number in their reproductive tract. Males may transfer accessory substances to protect their ejaculates against female selective processes, which benefits males but can harm females. In turn, females may use accessory gland fluids to control paternity or sperm storage. Female yellow dung flies (Scathophaga stercoraria) have paired accessory glands that produce fluids involved in fertilization and egg laying. One proposed function for these fluids is spermicide. Alternatively, female accessory gland fluid may help keep sperm alive to avoid fertilization failure or encourage sperm competition. Using yellow dung flies, we investigated the interaction of female accessory gland fluid with sperm in vitro. Significantly more sperm remained alive when exposed to accessory gland fluid compared to buffer only (63% vs. 44%). We conclude that female accessory gland fluid in yellow dung flies can help nourish rather than kill male sperm, although selective nourishment of sperm is as consistent with cryptic female choice as is selective spermicide.

A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and while its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo, using developing spermatogenic cells as a model. Remarkably, we find that AGO2 acts in the germ cell nucleus to positively regulate protein expression. We show that AGO2 dynamically binds both chromatin and nuclear mRNA transcripts of hundreds of genes required for sperm production, and germline conditional knockout (cKO) of Ago2 causes depletion of the corresponding proteins, along with defects in sperm number and morphology. Nuclear AGO2 partners with splicing, export, and chromatin factors to promote transcript export and protein expression. Together, our data reveal an unexpected role for nuclear AGO2 in enhancing expression of developmentally important genes.

Up-regulation of Arl4a gene expression by broccoli aqueous extract is associated with improved spermatogenesis in mouse testes

Introduction: Broccoli (Brassica oleracea) is well recognized due to its properties as an anti-cancer, antioxidant and scavenging free radicals. However, its benefit in enhancing spermatogenesis is not well understood. Objectives: To investigate the effect of broccoli aqueous extract on sperm factors and also expression of the involving genes (Catsper1, Catsper2, Arl4a, Sox5 and Sox9) in sperm factors in mice. Material and methods: Male mice were divided randomly into six groups: (1) Control, (2) Cadmium (3 mg/kg mouse body weight), (3) Orally treated with 200 broccoli aqueous extract (1 g ml-1), (4) Orally treated with 400 µl of broccoli aqueous extract, (5) Orally treated with 200 broccoli aqueous extract plus cadmium, and (6) Orally treated with 400 µl of broccoli aqueous extract plus cadmium. Sperms factors and also gene expression in Catsper1, Catsper2, Arl4a, Sox5 and Sox9 genes were studied. Results: An obvious improvement in sperm number and slight enhancement in sperm motility was observed in mice treated with broccoli extract with and without cadmium. While sperm viability was reduced by broccoli extract, except for 200 µl of broccoli extract with cadmium that was significantly increased. Interestingly, Arl4a gene expression showed an increase in 400 µl broccoli-treated group. Likewise, the Arl4a mRNA level in mice treated with cadmium along with 200 µl broccoli extract was higher than in cadmium-treated mice. Furthermore, broccoli extract enhanced the mRNA level of Catsper2 and Sox5 genes in mice treated with both 200 and 400 µl broccoli extract along with cadmium than the only cadmium-treated group. Conclusion: Generally, improvement in sperm count in broccoli-treated mice provides insight into the pharmaceutical industry to make new products available to infertile men.

P–070 Evaluation of SARS-CoV–2 in human semen and effect on total sperm number: A prospective observational study

Study question Is the SARS-CoV–2 virus present in human semen and what is the impact on semen parameters following an infection? Summary answer SARS-CoV–2 infection, though not detected in semen of recovered men, can affect TSN in ejaculate in the acute setting. What is known already Early epidemiological data has suggested that the primary mode of transmission is through respiratory droplets, but the presence of SARS-CoV–2 has been identified in other bodily fluids such as feces, urine, and semen. Study design, size, duration We prospectively recruited thirty men diagnosed with acute SARS-CoV–2 infection using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of pharyngeal swab specimens. Thirty semen samples from recovered men were obtained 11–64 days after testing positive for SAR-CoV–2 infection. The median duration between positive SAR-CoV–2 test and semen collection was 37 days (IQR=23). Participants/materials, setting, methods Semen samples were collected from each individual using mailed kits. Follow-up semen samples were done with mailed kits or in-person in office setting. Semen analysis and PCR was performed after samples were received. Main results and the role of chance The median total sperm number (TSN) in ejaculate was 12.5 million (IQR=53.1). When compared with age-matched SARS-CoV–2(-) men, TSN was lower among SARS-CoV–2(+) men (p = 0.0024). Five men completed a follow-up sperm analysis (median 3 months) and had a median TSN of 18 million (IQR=21.6). No RNA was detected by means of RT-PCR in the semen in 16 samples tested. Limitations, reasons for caution First, most of the semen samples came from non-severe men of whom were in the recovery stage and lacked symptoms. Additionally, our sample size was relatively small and overnight mail-in semen analysis kits were used during the acute phase of infection to minimize contact with positive subjects. Wider implications of the findings: Our findings suggest extremely low risk of viral transmission during sexual contact and assisted reproductive techniques, although further data need to be obtained. The impact on TSC in recovered men from SARS-CoV–2 infection is concerning, nevertheless long-term follow-up of these men is critical to determine the nadir of TSC. Trial registration number 20200401

O-150 Predicting the sperm retrieval of microsurgical testicular sperm extraction (mTESE) in infertile men with azoospermia on the day of TESE-ICSI

Study question Is the sperm retrieval rate of a small, pre-processed sample (PPS) of each TESE-biopsy representative for the sperm outcome on the day of ICSI? Summary answer The analysis of a PPS reliably reflects the probability of finding comparable numbers of sperm at time of TESE-ICSI. What is known already Azoospermia is defined as a condition where no spermatozoa are found in the ejaculate and is diagnosed in up to 15% among infertile men and in 11% of all patients attending our centre. The combination of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) has become the standard treatment of azoospermic patients. However, no validated standard procedure has been identified to predict the exact sperm outcome of the cryopreserved TESE samples prior to TESE-ICSI so far. For optimal management of TESE-biopsies and the respective ICSI treatment, we developed a stepwise approach for the analysis of tissue samples. Study design, size, duration We retrospectively analysed the outcome of 872 microsurgically retrieved testicular biopsies of 198 patients of legal age who had a TESE-ICSI at our department between 2009 and 2019. From all 872 mTESE biopsies the number of sperm extracted from a small, pre-processed sample (PPS) before freezing procedure were known. The PPS was then compared to the number of sperm retrieved from the corresponding thawed specimen on the day of TESE-ICSI. Participants/materials, setting, methods During micro-TESE eight samples per testis are retrieved, then 1/10 of each biopsy is removed, digested with collagenase and screened for spermatozoa (pre-processed sample, PPS). If less than 100 spermatozoa are detected the absolute sperm number is recorded, otherwise the result is displayed as the maximum value of 100 sperm. On the day of ICSI, one or more TESE biopsies are thawed and processed for TESE-ICSI; the absolute sperm number is counted again. Main results and the role of chance Comparing the sperm yield of 872 TESE samples at time of ICSI to its respective PPS showed a similar sperm outcome with a minor deviation of ± 5 spermatozoa in 73.6% of all biopsies. However, 12.9% of the specimen had less and 13.4% had more spermatozoa. A negative sperm retrieval in the initial PPS was confirmed in 93.1% (268/288). PPS with 1-4 spermatozoa had a 27.2% (43/158) risk of complete absence of sperm on the day of ICSI, yet sperm detection (≥1 sperm) was positive in 72.8% (115/158) of the biopsies. With initially ≥5 spermatozoa present in the PPS, only 0.9% (4/426) of the biopsies had no sperm on the day of ICSI, vice versa 99.1% (422/426) were spermatozoa positive. A significant (p = 0.01) and strong (rs = 0.926) correlation of the sperm retrieval rates of the PPS and the ICSI sample was found meaning that the PPS reflects very well the sperm retrieval rate of the cryopreserved mTESE biopsy thawed at time of TESE-ICSI. However, if ≤ 4 sperm are found in the PPS, there is a relevant risk for a negative sperm retrieval on the day of ICSI and the couple should be carefully advised before start of treatment. Limitations, reasons for caution This analysis focussed on sperm prediction in cases of severe male factor infertility and therefore the sperm yield on the day of ICSI was chosen as primary outcome. The reproductive competence of the retrieved sperm in terms of pregnancy and birth rates should be subject to further investigation. Wider implications of the findings Treatment options for azoospermic patients are mostly related to the ability to find sperm on the day of ICSI. However, validated standards for sperm processing are missing. Therefore, a PPS seems to be a good option for prediction of sperm retrieval and improves counselling of the patients prior to TESE-ICSI. Trial registration number not applicable

THE EFFECT OF CONSUMING AVOCADO (Persea americana) ON MICE (Mus musculus) SPERM QUALITY

The nutrients such as protein and vitamin are proven to improve the sperm quality. One nutrient rich fruit is avocado round green variety, which contains vitamins A, C and E higher than other varieties. This study aimed to determine the effect of consuming avocado on the mice sperm quality. This experimental study was using a Completely Randomized Design with four treatments, each with eight replications. Avocado dosage treatments were control, 75 % w/v, 100% w/v, and 133% w/v , with the number of mice were 32 age four weeks. Avocado was given three times a day, each 0.5 mL for six weeks besides the main food. The observed response was the sperm quality including number, motility and morphology. Sperm was taken from the epididymis after the mice were killed by cervical dislocation method. Sperm was made a suspension using 0.9% NaCl solution and a smear preparation to observe the sperm morphology while staining with basic stain crystal violet. The sperm quality was observed by Neubauer's counting rooms through a multimedia microscope. Data was analyzed using Kurskal Wallis test for the sperm number and Anova test for motility and morphological responses. The result showed that avocado had a significant influence for reproductive health, particularly for increasing spermatozoa quality, including concentration, motility and morphology of sperm. The higher dose of avocado given the higher quantity and quality of sperm resulted.