Understanding the fundamental reproductive biology of a species is the first step toward identifying parameters that are critical for reproduction and for the development of assisted reproductive techniques. Ejaculates were collected from aquarium (n = 24) and in situ (n = 34) sand tiger sharks Carcharias taurus. Volume, pH, osmolarity, sperm concentration, motility, status, morphology, and plasma membrane integrity were assessed for each ejaculate. Semen with the highest proportion of motile sperm was collected between April and June for both in situ and aquarium sand tiger sharks indicating a seasonal reproductive cycle. Overall, 17 of 30 semen samples collected from aquarium sharks from April through June contained motile sperm compared to 29 of 29 of in situ sharks, demonstrating semen quality differences between aquarium and in situ sharks. Sperm motility, status, morphology, and plasma membrane integrity were significantly higher (P < 0.05) for in situ compared to aquarium sand tiger sharks. Testosterone was measured by an enzyme immunoassay validated for the species. Testosterone concentration was seasonal for both aquarium and in situ sharks with highest concentrations measured in spring and lowest in summer. In situ sharks had higher (P < 0.05) testosterone concentration in spring than aquarium sharks. This study demonstrated annual reproduction with spring seasonality for male sand tiger sharks through marked seasonal differences in testosterone and semen production. Lower testosterone and poorer semen quality was observed in aquarium sharks likely contributing to the species' limited reproductive success in aquariums.

Summary sentence

During mating season, in situ sand tiger sharks Carcharias taurus have higher plasma testosterone and better semen quality than aquarium housed sand tiger sharks impairing reproductive success of aquarium populations.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.

Summary sentence

Testicular C. muridarum infection results in tissue damage and poor sperm quality and gives rise to offspring with serious abnormalities and developmental delays.

Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca⁺⁺ influxes while myeloperoxidase activity and as ERK1/2- and PI3Krelated signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

Summary Sentence

Confrontation of bovine PMN with sperm cell preparation results in NOX-, PAD4-, and Ca⁺⁺dependent NET formation.

Anti-Müllerian hormone (AMH) inhibits the activation of primordial follicles in the ovary. This causes an increased rate of ovarian reserve depletion in Amh^–/– mice. The depletion of the ovarian reserve is responsible for the onset of menopause but age-related infertility occurs in advance of ovarian reserve depletion. To determine whether accelerated loss of primordial follicles leads to earlier onset infertility, Amh^–/– and Amh^+/+ females were paired with Amh^+/+ stud males and birth rates were recorded across the females' reproductive lifespan. The number of primordial follicles remaining in the ovaries of Amh^–/– and Amh^+/+ females were quantified in two cohorts at 11–12 and 12–13 months of age. As expected, the ovarian reserve in the Amh^–/– females became depleted approximately 1 month earlier than Amh^+/+ females. However, no difference was observed in the cumulative number of births over the lifespan, nor were there any differences in mean littersize at any age. It is possible that the reproductive lifespan of mice is too short for sufficient divergence of primordial follicles numbers to cause differences in Amh^–/– and Amh^+/+ female fertility. An alternative explanation contradicts current thinking; the function of AMH may be unrelated to the longevity of the reproductive lifespan in female mice.

Summary sentence

Female Amh^–/– mice experience ovarian reserve depletion approximately one month earlier than Amh^+/+ females but experience no reduction in lifetime reproductive output.

The precise mechanisms of the reproductive physiological processes, such as labor initiation, are poorly understood. Oxytocin (OT) is one of the well-known uterotonics and is clinically adopted as a medication to facilitate childbirth. Vasopressin (VP), a posterior pituitary hormone similar to OT, has also been proposed to be involved in the reproductive physiology. In this study, we found that a total deficiency of V1a receptor subtype (V1aR) in mice resulted in a reduced number of pups, delayed labor initiation, and increased post-delivery hemorrhage compared with those in wild-type mice. Among the VP receptor subtypes, only V1aR was found to be expressed in the murine uterus, and its distribution pattern was different from that of the oxytocin receptor (OTR); V1aR expression was mainly distributed in the circular myometrium, whereas OTR was strongly expressed in both the circular and longitudinal myometrium. The maximum contractile force of the circular myometrium, induced by VP or OT, was attenuated in the pregnant uterus of Avpr1a-deficient mice. Contrarily, while OT expression was decreased in the Avpr1a-deficient uterus, OTR expression was significantly increased. These results suggest that V1aR deficiency not only reduces the uterine contractile force but also perturbs the expression of genes responsible for the reproductive physiology. Therefore, V1aR is necessary to exert the maximum contraction of the circular myometrium to deliver pups. This study revealed an important role of V1aR in physiological contraction and term parturition in mice.

Summary sentence

Vasopressin V1a receptor deficiency in mice resulted in fewer pups, delayed labor initiation, increased post-delivery hemorrhage, and attenuation of the maximum contractile force of the circular myometrium.

Uterine contractions are important for various functions of the female reproductive cycle. Contractions are generated, in part, by electrical coupling of smooth muscle cells of the myometrium, the main muscle layer of the uterus. Aberrant myometrial electrical activity can lead to uterine dysfunction. To better understand and treat conditions associated with aberrant activity, it is crucial to understand the mechanisms that underlie normal activity. Here, we used microelectrode array (MEA) to simultaneously record and characterize myometrial electrical activities at high spatial and temporal resolution. Mouse myometrial longitudinal muscle tissue was isolated at different stages throughout the estrous cycle and placed on an 8×8 MEA. Electrical activity was recorded for 10 min at a sampling rate of 12.5 kHz. We used a spike-tracking algorithm to independently analyze each channel and developed a pipeline to quantify the amplitude, duration, frequency, and synchronicity of the electrical activities. Electrical activities in estrous were more synchronous, and had shorter duration, higher frequency, and lower amplitude than electrical activities in non-estrous. We conclude that MEA can be used to detect differential patterns of myometrial electrical activity in distinct estrous cycle stages. In the future, this methodology can be used to assess different physiological and pathological states and evaluate therapeutic agents that regulate uterine function.

Endometriosis is a common estrogen-dependent inflammatory disease characterized by the presence of endometrial-like tissue outside the uterine cavity, which causes infertility and pelvic pain. Polymorphisms in MALAT1 have been demonstrated to play crucial roles in many diseases. However, the roles of MALAT1 polymorphisms in the etiology of endometriosis have not been well documented. We genotyped three MALAT1 polymorphisms in 555 endometriosis patients and 535 female control participants using quantitative polymerase chain reaction with TaqMan probes. To estimate the associations between MALAT1 polymorphisms and endometriosis risk, an unconditional logistic regression model was conducted to calculate an odds ratio (OR) and the 95% confidence interval (CI), adjusting for age, abortion history, number of deliveries, Body Mass Index (BMI), and The International Federation of Gynecology and Obstetrics (FIGO) stage. We found that the MALAT1 rs591291 C > T polymorphism significantly enhanced endometriosis risk (heterogeneous: adjusted OR = 1.36, 95% CI = 1.00–1.85, P = 0.050; homogenous: adjusted OR = 1.55, 95% CI = 1.03–2.33, P = 0.037; dominant: adjusted OR = 1.41, 95% CI = 1.05–1.88, P = 0.021). In stratification analyses, these associations were more predominant in the patients younger than 35 years old, with a relatively high number of deliveries and with a BMI between 25 and 29.9. Compared with wild-type CCG haplotype carriers, individuals with TCC haplotypes had a higher risk of developing endometriosis. The MALAT1 rs591291 C > T polymorphism was associated with a significant increase in endometriosis risk.

Summary sentence

We genotyped three MALAT1 polymorphisms (rs591291 C > T, rs664589 C > G, and rs4102217 G > C) in 555 endometriosis cases and 535 female controls using quantitative polymerase chain reaction with TaqMan probes to estimate the association between MALAT1 polymorphisms and the risk of endometriosis.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

Summary sentence

The tyrosine kinase receptor Ephb2 is required for normal proliferation and stem cell activity of adult mouse spermatogonia in long-term culture.

F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/β-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.

Summary sentence

Fbxw7 is essential for pubertal testis development and spermatogenesis by regulating SC maturation and function, potentially through degradation of GATA-4.

In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility.

Summary sentence

TESMIN protein, a member of metallothionein family, is essential for mouse spermatogenesis, especially in the meiotic stage.